Dynamic virulence-related regions of the plant pathogenic fungus Verticillium dahliae display enhanced sequence conservation
Depotter, Jasper R.L. ; Shi-Kunne, Xiaoqian ; Missonnier, Hélène ; Liu, Tingli ; Faino, Luigi ; Berg, Grardy C.M. van den; Wood, Thomas A. ; Zhang, Baolong ; Jacques, Alban ; Seidl, Michael F. ; Thomma, Bart P.H.J. - \ 2019
Molecular Ecology 28 (2019)15. - ISSN 0962-1083 - p. 3482 - 3495.
comparative genomics - effector - genome evolution - mutagenesis - two-speed genome - Verticillium wilt
Plant pathogens continuously evolve to evade host immune responses. During host colonization, many fungal pathogens secrete effectors to perturb such responses, but these in turn may become recognized by host immune receptors. To facilitate the evolution of effector repertoires, such as the elimination of recognized effectors, effector genes often reside in genomic regions that display increased plasticity, a phenomenon that is captured in the two-speed genome hypothesis. The genome of the vascular wilt fungus Verticillium dahliae displays regions with extensive presence/absence polymorphisms, so-called lineage-specific regions, that are enriched in in planta-induced putative effector genes. As expected, comparative genomics reveals differential degrees of sequence divergence between lineage-specific regions and the core genome. Unanticipated, lineage-specific regions display markedly higher sequence conservation in coding as well as noncoding regions than the core genome. We provide evidence that disqualifies horizontal transfer to explain the observed sequence conservation and conclude that sequence divergence occurs at a slower pace in lineage-specific regions of the V. dahliae genome. We hypothesize that differences in chromatin organisation may explain lower nucleotide substitution rates in the plastic, lineage-specific regions of V. dahliae.
Virulence contribution and recognition of homologs of the Verticillium dahliae effector Ave1
Boshoven, Jordi C. - \ 2017
Wageningen University. Promotor(en): B.P.H.J. Thomma; P.J.G.M. de Wit. - Wageningen : Wageningen University - ISBN 9789463436441 - 183
verticillium dahliae - plant pathogenic fungi - plant pathogens - disease resistance - virulence factors - virulence - immunity - host parasite relationships - plant-microbe interactions - symbiosis - mutagenesis - resistance breeding - verticillium dahliae - plantenziekteverwekkende schimmels - plantenziekteverwekkers - ziekteresistentie - virulente factoren - virulentie - immuniteit - gastheer parasiet relaties - plant-microbe interacties - symbiose - mutagenese - resistentieveredeling
Disease resistance in crops is an important aspect of securing global food security. Resistant plants carry immune receptors that sense pathogen invasion often through the recognition of important pathogen virulence factors, known as effectors. Thus, identification and characterization of effectors is important for the fundamental understanding of virulence mechanisms and to aid in resistance breeding. In this thesis the VdAve1 effector of the soil-borne fungal pathogen Verticillium dahliae is studied that is recognized by tomato immune receptor Ve1. Homologs were found in other plant pathogens and the role in virulence in these pathogens was analyzed. Ave1 homologs are differentially recognized by Ve1 and with a combination of domain swaps and truncations a surface exposed patch was identified that contributes to the recognition by Ve1. Knowledge of specific effector-receptor combinations and knowledge of effectors in general can be exploited to aid in breeding for durable resistance in crops.
Transcriptional and functional targets of SCHIZORIZA in root development
Liere, Sabine van - \ 2017
Wageningen University. Promotor(en): B.J.G. Scheres, co-promotor(en): R. Heidstra. - Wageningen : Wageningen University - ISBN 9789463437998 - 122
arabidopsis thaliana - biological development - root meristems - root caps - cell division - stem cells - transcriptomics - gene regulation - mutagenesis - arabidopsis thaliana - biologische ontwikkeling - wortelmeristemen - wortelmutsjes - celdeling - stamcellen - transcriptomica - genregulatie - mutagenese
In this thesis I focus on SCHIZORIZA, a gene involved in tissue specification and cell fate segregation in the Arabidopsis root. Chapter 1 describes asymmetric cell division, Arabidopdis embryo development and root meristem development. In more detail we describe the maintenance of quiescent centre and columella stem cells, the development of ground tissue and epidermis/ lateral root cap. Finally we introduce SCHIZORIZA (SCZ) as a factor involved in radial patterning and the maintenance of cortex identity.
In Chapter 2, we study the interaction between the SCZ and SHORTROOT/ SCARECROW pathways that are required in parallel during stem cell niche specification in embryogenesis for the maintenance of tissue fates. Here we investigate the strong synergy of shr and scz mutants and show that at late torpedo stage scz;shr double mutant embryos lose both ground tissue and meristem marker expression.
Chapter 3 describes the use of a transcriptomics approach to identify genes differentially regulated by SCZ. These differentially regulated genes can be divided into two distinct tissue enriched groups. Upregulated genes are enriched for root cap expression and cortex expressed genes are overrepresented in the downregulated set of genes. A subset of the upregulated genes has a HSE associated with their promoter and therefore possibly represents direct SCZ targets.
In Chapter 4 we describe a mutagenesis screen to identify functional downstream targets of SCZ. Using a cortex and lateral root cap tissue marker, we identified two suppressors of the scz mutant. Both restore the fate segregation phenotype of scz mutants. We used whole genome deep sequencing to map the causal suppressor mutations in the LBD12 gene.
The analysis of LBD12 function is described in Chapter 5. We show that the single lbd12 mutant has a QC and columella phenotype. In addition, we show that ectopic expression of LBD12 induces ectopic divisions.
Dissecting hormonal pathways in nitrogen-fixing rhizobium symbioses
Zeijl, Arjan van - \ 2017
Wageningen University. Promotor(en): T. Bisseling, co-promotor(en): R. Geurts. - Wageningen : Wageningen University - ISBN 9789463436311 - 231
plants - root nodules - rhizobium - symbiosis - cytokinins - plant-microbe interactions - biosynthesis - mutagenesis - genes - nodulation - planten - wortelknolletjes - rhizobium - symbiose - cytokininen - plant-microbe interacties - biosynthese - mutagenese - genen - knobbelvorming
Nitrogen is a key element for plant growth. To meet nitrogen demands, some plants establish an endosymbiotic relationship with nitrogen-fixing rhizobium or Frankia bacteria. This involves formation of specialized root lateral organs, named nodules. These nodules are colonized intracellularly, which creates optimal physiological conditions for the fixation of atmospheric nitrogen by the microbial symbiont. Nitrogen-fixing endosymbioses are found among four related taxonomic orders that together form the nitrogen-fixation clade. Within this clade, nodulation is restricted to ten separate lineages that are scattered among mostly non-nodulating plant species. This limited distribution suggests that genetic adaptations that allowed nodulation to evolve occurred in a common ancestor.
A major aim of the scientific community is to unravel the evolutionary trajectory towards a nitrogen-fixing nodule symbiosis. The formation of nitrogen-fixing root nodules is best studied in legumes (Fabaceae, order Fabales); especially in Lotus japonicus and Medicago truncatula, two species that serve as model. Legumes and Parasponia (Cannabaceae, order Rosales) represent the only two lineages that can form nodules with rhizobium bacteria. Studies on M. truncatula, L. japonicus and Parasponia showed, amongst others, that nodule formation is initiated upon perception of rhizobial secreted lipo-chitooligosaccharide (LCO) signals. These signals are structurally related to the symbiotic signals produced by arbuscular mycorrhizal fungi. These obligate biotropic fungi colonize roots of most land plants and form dense hyphal structures inside existing root cortical cells.
Rhizobial and mycorrhizal LCOs are perceived by LysM-domain-containing receptor-like kinases. These activate a signaling pathway that is largely shared between both symbioses. Symbiotic LCO receptors are closely related to chitin innate immune receptors, and some receptors even function in symbiotic as well as innate immune signaling. In Chapter 2, I review the intertwining of symbiotic LCO perception and chitin-triggered immunity. Furthermore, I discuss how rhizobia and mycorrhiza might employ LCO signaling to modulate plant immunity. In a perspective, I speculate on a role for plant hormones in immune modulation, besides an important function in nodule organogenesis.
In legumes, nodule organogenesis requires activation of cytokinin signaling. Mutants in the orthologous cytokinin receptor genes MtCRE1 and LjLHK1 in M. truncatula and L. japonicus, respectively, are severely affected in nodule formation. However, how cytokinin signaling is activated in response to rhizobium LCO perception and to what extent this contributes to rhizobium LCO-induced signaling remained elusive. In Chapter 3, I show that the majority of transcriptional changes induced in wild-type M. truncatula, upon application of rhizobium LCOs, are dependent on activation of MtCRE1-mediated cytokinin signaling. Among the genes induced in wild type are several involved in cytokinin biosynthesis. Consistently, cytokinin measurements indicate that cytokinins rapidly accumulate in M. truncatula roots upon treatment with rhizobium LCOs. This includes the bioactive cytokinins isopentenyl adenine and trans-zeatin. Therefore, I argue that cytokinin accumulation represents a key step in the pathway leading to legume root nodule organogenesis.
Strigolactones are plant hormones of which biosynthesis is increased in response to nutrient limitation. In rice (Oryza sativa) and M. truncatula, this response requires the GRAS-type transcriptional regulators NSP1 and NSP2. Both proteins regulate expression of DWARF27 (D27), which encodes an enzyme that performs the first committed step in strigolactone biosynthesis. NSP1 and NSP2 are also essential components of the signaling cascade that controls legume root nodule formation. In line with this, I questioned whether the NSP1-NSP2-D27 regulatory module functions in rhizobium symbiosis. In Chapter 4, I show that in M. truncatula MtD27 expression is induced within hours after treatment with rhizobium LCOs. Spatiotemporal expression studies revealed that MtD27 is expressed in the dividing cells of the nodule primordium. At later stages, its expression becomes confined to the meristem and distal infection zone of the mature nodule. Analysis of the expression pattern of MtCCD7 and MtCCD8, two additional strigolactone biosynthesis genes, showed that these genes are co-expressed with MtD27 in nodule primordia and mature nodules. Additionally, I show that symbiotic expression of MtD27 requires MtNSP1 and MtNSP2. This suggests that the NSP1-NSP2-D27 regulatory module is co-opted in rhizobium symbiosis.
Comparative studies between legumes and nodulating non-legumes could identify shared genetic networks required for nodule formation. We recently adopted Parasponia, the only non-legume lineage able to engage in rhizobium symbiosis. However, to perform functional studies, powerful reverse genetic tools for Parasponia are essential. In Chapter 5, I describe the development of a fast and efficient protocol for CRISPR/Cas9-mediated mutagenesis in Agrobacterium tumefaciens-transformed Parasponia andersonii plants. Using this protocol, stable mutants can be obtained in a period of three months. These mutants can be effectively propagated in vitro, which allows phenotypic evaluation already in the T0 generation. As such, phenotypes can be obtained within six months after transformation. As proof-of-principle, we mutated PanHK4, PanEIN2, PanNSP1 and PanNSP2. These genes are putatively involved in cytokinin and ethylene signaling and regulation of strigolactone biosynthesis, respectively. Additionally, orthologues of these genes perform essential symbiotic functions in legumes. Panhk4 and Panein2 knockout mutants display developmental phenotypes associated with reduced cytokinin and ethylene signaling. Analysis of Pannsp1 and Pannsp2 mutants revealed a conserved role for NSP1 and NSP2 in regulation of the strigolactone biosynthesis genes D27 and MAX1 and root nodule organogenesis. In contrast, symbiotic mutant phenotypes of Panhk4 and Panein2 mutants are different from their legume counterparts. This illustrates the value of Parasponia as comparative model - besides legumes - to study the genetics underlying rhizobium symbiosis.
Phylogenetic reconstruction showed that the Parasponia lineage is embedded in the non-nodulating Trema genus. This close relationship suggests that Parasponia and Trema only recently diverged in nodulation ability. In Chapter 6, I exploited this close relationship to question whether the nodulation trait is associated with gene expression differentiation. To this end, I sequenced root transcriptomes of two Parasponia and three Trema species. Principal component analysis separated all Parasponia samples from those of Trema along the first principal component. This component explains more than half of the observed variance, indicating that the root transcriptomes of two Parasponia species are distinct from that of the Trema sister species T. levigata, as well as the outgroup species T. orientalis and T. tomentosa. To determine, whether the transcriptional differences between Parasponia and Trema are relevant in a symbiotic context, I compared the list of differentially expressed genes to a list of genes that show nodule-enhanced expression in P. andersonii. This revealed significant enrichment of nodule-enhanced genes among genes that lower expressed in roots of Parasponia compared to Trema. Among the genes differentially expressed between Parasponia and Trema roots are several involved in mycorrhizal symbiosis as well as jasmonic acid biosynthesis. Measurements of hormone concentrations, showed that Parasponia and Trema roots harbor a difference in jasmonic acid/salicylic acid balance. However, mutants in jasmonic acid biosynthesis are unaffected in nodule development. Therefore, it remains a challenge to determine whether the difference in root transcriptomes between Parasponia and Trema are relevant in a symbiotic context.
In Chapter 7, I review hormone function in nitrogen-fixing nodule symbioses in legumes, Parasponia and actinorhizal species. In this chapter, I question whether different nodulating lineages recruited the same hormonal networks to function in nodule formation. Additionally, I discuss whether nodulating species harbor genetic adaptations in hormonal pathways that correlate with nodulation capacity.
Opportunities of New Plant Breeding Techniques
Schaart, Jan ; Riemens, M.M. ; Wiel, C.C.M. van de; Lotz, L.A.P. ; Smulders, M.J.M. - \ 2015
Wageningen : Wageningen UR - 24
plantenveredeling - plantenveredelingsmethoden - resistentieveredeling - cisgenese - intragenic recombination - mutagenese - dna-methylering - bloei - plant breeding - plant breeding methods - resistance breeding - cisgenesis - intragenic recombination - mutagenesis - dna methylation - flowering
This brochure gives an overview of new plant breeding techniques. This overview is based on a more technical review of the scientific literature, published in a separate report. The overview presents the opportunities and limitations of these techniques from the point of view of potential applications in plant breeding with promising results for improving agricultural sustainability.
Genetic resources for quantitative trait analysis: novelty and efficiency in design from an Arabidopsis perspective
Wijnen, C.L. ; Keurentjes, J.J.B. - \ 2014
Current Opinion in Plant Biology 18 (2014). - ISSN 1369-5266 - p. 103 - 109.
genome-wide association - inbred line population - functional genomics - thaliana accession - induced mutations - natural variation - identification - plant - mutagenesis - phenotypes
The use of genetic resources for the analysis of quantitative traits finds its roots in crop breeding but has seen a rejuvenation in Arabidopsis thaliana thanks to specific tools and genomic approaches. Although widely used in numerous crop and natural species, many approaches were first developed in this reference plant. We will discuss the scientific background and historical use of mapping populations in Arabidopsis and highlight the technological innovations that drove the development of novel strategies. We will especially lay emphasis on the methodologies used to generate the diverse population types and designate possible applications. Finally we highlight some of the most recent developments in generating genetic mapping resources and suggest specific usage for these novel tools and concepts.
Dominant negative phenotype of Bacillus thuringiensis Cry1Ab, Cry11Aa and Cry4Ba mutants suggest hetero-oligomer formation among different Cry toxins.
Carmona, D. ; Rodriguez-Almazan, C. ; Munoz-Garay, C. ; Portugal, L. ; Perez, C. ; Maagd, R.A. de; Bakker, P. ; Soberon, M. ; Bravo, A. - \ 2011
PLoS ONE 6 (2011)5. - ISSN 1932-6203 - 6 p.
subsp israelensis - delta-endotoxin - manduca-sexta - alkaline-phosphatase - protective antigen - anopheles-gambiae - toxicity - crystal - anthrax - mutagenesis
Background - Bacillus thuringiensis Cry toxins are used worldwide in the control of different insect pests important in agriculture or in human health. The Cry proteins are pore-forming toxins that affect the midgut cell of target insects. It was shown that non-toxic Cry1Ab helix a-4 mutants had a dominant negative (DN) phenotype inhibiting the toxicity of wildtype Cry1Ab when used in equimolar or sub-stoichiometric ratios (1:1, 0.5:1, mutant:wt) indicating that oligomer formation is a key step in toxicity of Cry toxins. Methodology/Principal Findings - The DN Cry1Ab-D136N/T143D mutant that is able to block toxicity of Cry1Ab toxin, was used to analyze its capacity to block the activity against Manduca sexta larvae of other Cry1 toxins, such as Cry1Aa, Cry1Ac, Cry1Ca, Cry1Da, Cry1Ea and Cry1Fa. Cry1Ab-DN mutant inhibited toxicity of Cry1Aa, Cry1Ac and Cry1Fa. In addition, we isolated mutants in helix a-4 of Cry4Ba and Cry11Aa, and demonstrate that Cry4Ba-E159K and Cry11Aa-V142D are inactive and completely block the toxicity against Aedes aegypti of both wildtype toxins, when used at sub-stoichiometric ratios, confirming a DN phenotype. As controls we analyzed Cry1Ab-R99A or Cry11Aa-E97A mutants that are located in helix a-3 and are affected in toxin oligomerization. These mutants do not show a DN phenotype but were able to block toxicity when used in 10:1 or 100:1 ratios (mutant:wt) probably by competition of binding with toxin receptors. Conclusions/Significance - We show that DN phenotype can be observed among different Cry toxins suggesting that may interact in vivo forming hetero-oligomers. The DN phenotype cannot be observed in mutants affected in oligomerization, suggesting that this step is important to inhibit toxicity of other toxins
HrcA and DnaK are important for static and continuous flow biofilm formation and disinfectant resistance in Listeria monocytogenes
Veen, S. van der; Abee, T. - \ 2010
Microbiology 156 (2010)12. - ISSN 1350-0872 - p. 3782 - 3790.
food-processing environment - heat-shock genes - benzalkonium chloride - streptococcus-mutans - sos response - egd-e - expression - operon - disaggregation - mutagenesis
The food-borne pathogen Listeria monocytogenes is able to form biofilms in food processing environments. Since biofilms are generally difficult to eradicate during clean-up procedures, they pose a major risk for the food industry. Stress resistance mechanisms involved in L. monocytogenes biofilm formation and disinfectant resistance have, to our knowledge, not been identified thus far. In this study, we investigated the role of hrcA, which encodes the transcriptional regulator of the class I heat-shock response, and dnaK, which encodes a class I heat-shock response chaperone protein, in static and continuous-flow biofilm formation and resistance against benzalkonium chloride and peracetic acid. Induction of both hrcA and dnaK during continuous-flow biofilm formation was observed using quantitative real-time PCR and promoter reporters. Furthermore, in-frame deletion and complementation mutants of hrcA and dnaK revealed that HrcA and DnaK are required to reach wild-type levels of both static and continuous-flow biofilms. Finally, disinfection treatments of planktonic-grown cells and suspended static and continuous-flow biofilm cells of wild-type and mutants showed that HrcA and DnaK are important for resistance against benzalkonium chloride and peracetic acid. In conclusion, our study revealed that HrcA and DnaK are important for L. monocytogenes biofilm formation and disinfectant resistance
A universal approach to eliminate antigenic properties of alpha-gliadin peptides in celiac disease
Mitea, C. ; Salentijn, E.M.J. ; Veelen, P. van; Goryunova-Svetlana, V. ; Meer, I.M. van der; Broeck, H.C. van den; Mujico, J.R. ; Monserrat, V. ; Gilissen, L.J.W.J. ; Drijfhout, J.W. ; Dekking, L. ; Smulders, M.J.M. - \ 2010
PLoS ONE 5 (2010). - ISSN 1932-6203 - 12 p.
t-cell response - tissue transglutaminase - cereal toxicity - wheat - epitopes - arabidopsis - mutagenesis - prevalence - varieties - explains
Celiac disease is caused by an uncontrolled immune response to gluten, a heterogeneous mixture of wheat storage proteins, including the a-gliadins. It has been shown that a-gliadins harbor several major epitopes involved in the disease pathogenesis. A major step towards elimination of gluten toxicity for celiac disease patients would thus be the elimination of such epitopes from a-gliadins. We have analyzed over 3,000 expressed a-gliadin sequences from 11 bread wheat cultivars to determine whether they encode for peptides potentially involved in celiac disease. All identified epitope variants were synthesized as peptides and tested for binding to the disease-associated HLA-DQ2 and HLA-DQ8 molecules and for recognition by patient-derived a-gliadin specific T cell clones. Several specific naturally occurring amino acid substitutions were identified for each of the a-gliadin derived peptides involved in celiac disease that eliminate the antigenic properties of the epitope variants. Finally, we provide proof of principle at the peptide level that through the systematic introduction of such naturally occurring variations a-gliadins genes can be generated that no longer encode antigenic peptides. This forms a crucial step in the development of strategies to modify gluten genes in wheat so that it becomes safe for celiac disease patients. It also provides the information to design and introduce safe gluten genes in other cereals, which would exhibit improved quality while remaining safe for consumption by celiac disease patients.
Dependence of continuous-flow biofilm formation by Listeria monocytogenes EGD-e on SOS response factor YneA
Veen, S. van der; Abee, T. - \ 2010
Applied and Environmental Microbiology 76 (2010)6. - ISSN 0099-2240 - p. 1992 - 1995.
dna-polymerase - cell-division - attachment - mutagenesis - protein
Listeria monocytogenes was previously shown to form biofilms composed of a network of knitted chains under continuous-flow conditions. Here we show that the SOS response is activated under these conditions and that deletion of its regulon member yneA results in diminished biofilm formation under continuous-flow conditions
Nucleases Encoded by Integraded Elements CJIE2 and CJIE4 Inhibit Natural Transformation of Campylobacter Jejuni
Gaasbeek, E.J. ; Wagenaar, J.A. ; Guilhabert, M.R. ; Putten, J.P. van; Parker, C.T. ; Wal, F.J. van der - \ 2010
Journal of Bacteriology 192 (2010)4. - ISSN 0021-9193 - p. 936 - 941.
serratia-marcescens endonuclease - horizontal gene-transfer - diversity - bacteria - sequence - dnase - identification - mutagenesis - mechanism - strains
The species Campylobacter jejuni is naturally competent for DNA uptake; nevertheless, nonnaturally transformable strains do exist. For a subset of strains we previously showed that a periplasmic DNase, encoded by dns, inhibits natural transformation in C. jejuni. In the present study, genetic factors coding for DNase activity in absence of dns were identified. DNA arrays indicated that nonnaturally transformable dns-negative strains contain putative DNA/RNA non-specific endonucleases encoded by CJE0566 and CJE1441 of strain RM1221. These genes are located on C. jejuni integrated element 2 and 4. Expression of CJE0566 and CJE1441 from strain RM1221 and a homologous gene from strain 07479 in DNase-negative Escherichia coli and C. jejuni strains indicated that these genes code for DNases. Genetic transfer of the genes to a naturally transformable C. jejuni strain resulted in a decreased efficiency of natural transformation. Modelling suggests that the C. jejuni DNases belong to the Serratia nuclease family. Overall, the data indicate that the acquisition of prophage encoded DNA/RNA non-specific endonucleases inhibits the natural transformability of C. jejuni through hydrolysis of DNA
The crystal structure of a hyperthermoactive exopolygalacturonase from Thermotoga maritima reveals a unique tetramer
Pijning, T. ; Pouderoyen, G. van; Kluskens, L.D. ; Oost, J. van der; Dijkstra, B.W. - \ 2009
FEBS Letters 583 (2009)22. - ISSN 0014-5793 - p. 3665 - 3670.
active-site - endopolygalacturonase-i - aspergillus-aculeatus - sequence alignments - polygalacturonase - protein - features - crystallography - mutagenesis - family-28
The exopolygalacturonase from Thermotoga maritima is the most thermoactive and thermostable pectinase known to date. Here we present its crystal structure at 2.05A resolution. High structural homology around the active site allowed us to propose a model for substrate binding, explaining the exo-cleavage activity and specificity for non-methylated saturated galacturonate at the non-reducing end. Furthermore, the structure reveals unique features that contribute to the formation of stable tetramers in solution. Such an oligomerization has not been observed before for polygalacturonases
On the natural and laboratory evolution of an antibiotic resistance gene
Salverda, M.L.M. - \ 2008
Wageningen University. Promotor(en): Rolf Hoekstra, co-promotor(en): Arjan de Visser; John van der Oost. - [S.l.] : S.n. - ISBN 9789085049999 - 144
evolutie - bèta-lactamase - plasmiden - recombinatie - bacteriën - fenotypen - mutagenese - fylogenie - moleculaire genetica - selectie - antibioticaresistentie - evolution - beta-lactamase - plasmids - recombination - bacteria - phenotypes - mutagenesis - phylogeny - molecular genetics - selection - antibiotic resistance
TEM-1 ß-lactamase is one of the most notorious antibiotic resistance enzymes around. It exists at high frequencies in antibiotic-resistant bacteria around the world and confers resistance to ß-lactam antibiotics, including penicillins (e.g. ampicillin) and cephalosporins. The enzyme displays a remarkable phenotypic plasticity in response to the introduction of new drugs; within a few years after the clinical debut of most new ß-lactam antibiotics resistance conferring variants of TEM-1 are isolated. Such a shift in resistance phenotype is typically caused by just a few amino acid substitutions. Until today, more than 150 variants of TEM-1 with a unique amino acid sequence have been identified.
Because of the clear link between genotype and phenotype (i.e. level of resistance or fitness) and because of the ease of selecting for increased antibiotic resistance, TEM-1 has been used as a model in studies that seek new methods to optimize proteins. These studies combine the power of in vitro mutagenesis and in vivo selection and have resulted in a wealth of information about which mutations can increase resistance when the enzyme is exposed to an antibiotic that it initially hydrolyzes inefficiently. At a later stage, these techniques were adopted and used to repeat and predict the natural evolution of TEM-1 under various selective conditions. Recently, TEM-1 is increasingly being used as an experimental model for the study of fundamental evolutionary questions, particularly those that benefit from the direct relationship between genotype and phenotype.
In this thesis, both the natural and laboratory evolution of TEM-1 are studied. The aim of the laboratory work is to increase our understanding of the way in which adaptive mutations interact. For this purpose, TEM-1 is mutagenized using error-prone PCR, which creates variation in the resulting copies of the TEM-1 gene. Mutated gene-copies are placed in bacteria which are subsequently selected for increased resistance to cefotaxime (an antibiotic that TEM-1 hydrolyzes poorly). By repeating this process multiple times in independent experiments, the mutations and mutational trajectories involved in the increase of cefotaxime resistance are studied. At a fundamental level, this has lead to a better understanding of the nature of mutation interaction and its consequences for evolutionary contingency and constraint. Evidence indicating that certain ‘silent’ mutations (i.e. mutations that alter the codon sequence but not the amino acid that the respective codon encodes) can also play a role in increased resistance was found in these data as well.
A phylogenetic study of the sequences of the ~150 different TEM-alleles that have been isolated in hospitals and clinics so far indicates that recombination has played a significant role in the evolution of TEM-alleles, contrary to what is often assumed. Furthermore, amino acid substitutions present in these clinical isolates are compared to those found in laboratory evolution studies of TEM-1, in order to investigate to what extent laboratory evolution can be used as a predictive tool for the natural evolution of antibiotic resistance genes. This overview indicates that laboratory evolution very accurately repeats the natural evolution of TEM-1. Based on these findings, predictions are made about substitutions that may appear in future clinical TEM-isolates, and directions are given how laboratory evolution can be exploited as a predictive tool most efficiently.
EU-OSTID: A collection of transposon insertional mutants for functional genomics in rice
Enckevort, L.J.G. van; Droc, G. ; Piffanelli, P. ; Greco, R. ; Gagneur, C. ; Weber, C. ; Gonzalez, V.M. ; Cabot, P. ; Fornara, F. ; Berri, S. ; Miro, B. ; Lan, P. ; Rafel, M. ; Capell, T. ; Puigdomenech, P. ; Ouwerkerk, P.B.F. ; Meijer, A.H. ; Pe', E. ; Colombo, L. ; Christou, P. ; Guiderdoni, E. - \ 2005
Plant Molecular Biology 59 (2005)1. - ISSN 0167-4412 - p. 99 - 110.
reverse genetics - homeobox genes - mutagenesis - expression - system - family - annotation - generation - resources - activator
A collection of 1373 unique flanking sequence tags (FSTs), generated from Ac/Ds and Ac transposon lines for reverse genetics studies, were produced in japonica and indica rice, respectively. The Ds and Ac FSTs together with the original T-DNAs were assigned a position in the rice genome sequence represented as assembled pseudomolecules, and found to be distributed evenly over the entire rice genome with a distinct bias for predicted gene-rich regions. The bias of the Ds and Ac transposon inserts for genes was exemplified by the presence of 59% of the inserts in genes annotated on the rice chromosomes and 41% present in genes transcribed as disclosed by their homology to cDNA clones. In a screen for inserts in a set of 75 well annotated transcription factors, including homeobox-containing genes, we found six Ac/Ds inserts. This high frequency of Ds and Ac inserts in genes suggests that saturated knockout mutagenesis in rice using this strategy will be efficient and possible with a lower number of inserts than expected. These FSTs and the corresponding plant lines are publicly available through OrygenesDB database and from the EU consortium members.
Characterisation of Aspergillus niger prolyl aminopeptidase
Basten, E.J.W. ; Moers, A.P.H.A. ; Ooyen, A.J.J. van; Schaap, P.J. - \ 2005
Molecular Genetics and Genomics 272 (2005)6. - ISSN 1617-4615 - p. 673 - 679.
proline iminopeptidase gene - subsp bulgaricus cnrz-397 - campestris pv. citri - oligopeptidase family - serratia-marcescens - bacillus-coagulans - expressed enzyme - cloning - identification - mutagenesis
We have cloned a gene (papA) that encodes a prolyl aminopeptidase from Aspergillus niger. Homologous genes are present in the genomes of the Eurotiales A. nidulans, A. fumigatus and Talaromyces emersonii, but the gene is not present in the genome of the yeast Saccharomyces cerevisiae. Cell extracts of strains overexpressing the gene under the control of its own promoter showed a fourfold to sixfold increase in prolyl aminopeptidase activity, but no change in phenylalanine or leucine aminopeptidase activity. The overexpressed enzyme was subsequently purified and characterised. The enzyme specifically removes N-terminal proline and hydroxyproline residues from peptides. It is the first enzyme of its kind from a eukaryotic organism that has been characterised
Functional analysis of the putative fusion domain of the Baculovirus envelope fusion protein F
Westenberg, M. ; Veenman, F. ; Roode, E.C. ; Goldbach, R.W. ; Vlak, J.M. - \ 2004
Journal of Virology 78 (2004)13. - ISSN 0022-538X - p. 6946 - 6954.
californica multicapsid nucleopolyhedrovirus - membrane-fusion - genome sequence - cell-fusion - glycoprotein - virus - gp64 - peptide - site - mutagenesis
Group II nucleopolyhedroviruses (NPVs), e.g., Spodoptera exigua MNPV, lack a GP64-like protein that is present in group I NPVs but have an unrelated envelope fusion protein named F. In contrast to GP64, the F protein has to be activated by a posttranslational cleavage mechanism to become fusogenic. In several vertebrate viral fusion proteins, the cleavage activation generates a new N terminus which forms the so-called fusion peptide. This fusion peptide inserts in the cellular membrane, thereby facilitating apposition of the viral and cellular membrane upon sequential conformational changes of the fusion protein. A similar peptide has been identified in NPV F proteins at the N terminus of the large membrane-anchored subunit F-1. The role of individual amino acids in this putative fusion peptide on viral infectivity and propagation was studied by mutagenesis. Mutant F proteins with single amino acid changes as well as an F protein with a deleted putative fusion peptide were introduced in gp64-null Autographa californica MNPV budded viruses (BVs). None of the mutations analyzed had an major effect on the processing and incorporation of F proteins in the envelope of BVs. Only two mutants, one with a substitution for a hydrophobic residue (F152R) and one with a deleted putative fusion peptide, were completely unable to rescue the gp64-null mutant. Several nonconservative substitutions for other hydrophobic residues and the conserved lysine residue had only an effect on viral infectivity. In contrast to what was expected from vertebrate virus fusion peptides, alanine substitutions for glycines did not show any effect.
Functional genomics strategies with transposons in rice
Greco, R. - \ 2003
Wageningen University. Promotor(en): Maarten Koornneef, co-promotor(en): A.B. Pereira. - Wageningen : Wageningen Universiteit - ISBN 9789058089168 - 182
rijst - oryza - genexpressie - transposons - transpositie - transcriptie - mutagenese - genexpressieanalyse - rice - oryza - gene expression - transposable elements - transposition - transcription - mutagenesis - genomics
Rice is a major staple food crop and a recognizedmonocotylenedousmodel plant from which gene function discovery is projected to contribute to improvements in a variety of cereals like wheat and maize. The recent release of rough drafts of the rice genome sequence for public research provides a vast resource of gene sequences whose functions need to be determined by reverse genetics methods.Characterisation of a mutant phenotype is one of the most promising approaches to link gene to function. Based on this assumption, mutagenesis with transposable elements was chosen as a strategy in the research described in this thesis to address gene function in rice (Chapter 2). The well characterized maize Ac/Ds and En/Spm transposon systems were employed asinsertionalmutagens based on their known ability to transpose inheterologousspecies. Transposon insertions can cause a knockout mutation by blocking the correct expression of a gene, which may result in a mutant phenotype. The mutant gene, thus "tagged" by the inserted transposon, can then be isolated by recovering the DNA flanking the insert and lead to the isolation of the wild-type gene. Constructs for knockout mutagenesis were generated which employed the autonomous Ac element and were tested in rice japonica (Chapter 3) andindica(Chapter 4) varieties. However, the utility of knockout mutations is limited, as the majority of them display no obvious phenotype. This may be due to functional redundancy, where one or more other genes can substitute for the same function, to subtle and conditional phenotypes, or to early lethality caused by the mutation. Gene detection strategies have therefore been developed in addition to classical knockout mutagenesis to address the function of genes that do not directly reveal an obvious phenotype when disrupted (Chapter 5). To utilize gene detection, japonica rice was transformed with advanced two-component enhancer trap vectors, consisting of a mobile transposon element ( Ds or I/dSpm ), and the corresponding stabletransposase( Ac or En/Spm ) source under control of theCaMV35S promoter. The mobile transposons contained in this case a GUS marker gene driven by aCaMV35S minimal promoter that could display the pattern of expression of the adjacent trapped gene and thus provide a clue for its function (Chapters 6 and 7). A large number of rice transformants were produced to test the activity of the different transposon constructs, with the final aim of identifying optimal "starter" lines for the development of tagging populations. Among the factors evaluated were the propensity for continuous transposition through successive generations, the ability to generate large numbers of independent inserts in progeny plants and the target-site specificity of insertion. The usefulness of the selectable markers incorporated in the constructs was also assessed.The results revealed high mobility of the Ac/Ds system in rice (Chapter 3, 4 and 6), although inactivation of Ds was observed in later generations (from T 2 onward). Nevertheless, the high frequency of independent transposition demonstrated to occur in early generations (T 0 and T 1 ) enabled the production of T 2 and T 3 lines with independent "stabilised insertions, which can be used directly for reverse genetics screenings without further need for selection against thetransposasesource. The autonomous Ac transposon, in contrast, does not seem to lose mobility and was shown to efficiently transpose in japonica andindicagenotypes as well, supporting its further use in the establishment of a tagging system in this economically important subspecies. Both Ac and Ds displayed amplification of copy number, which enabled the generation of lines containing multiple transposons. Pilot sequencing of genomic sites flanking the Ac and Ds inserts revealed a preferential insertion of these transposons into genes or gene-rich regions and confirmed their tendency to transpose to linked sites, which makes them suitable for targeted tagging. Preliminary testing of the Ac/Ds enhancer trap lines for their ability to function as "detectors" of gene activity, revealed a low frequency of GUS staining patterns in somatic sectors. More thorough screenings are currently under way to fully evaluate the functionality of the system.In contrast to Ac/Ds , the En/Spm system displayed a surprisingly low transposition activity in rice (Chapter 7), restricted to somatic events that were not transmitted to the next generation, in spite of being a well-established mutagenic system inheterologousdicotyledonous species such as Arabidopsis. Transcription analysis of the En/Spm maize element in rice revealed that correct splicing of the element occurs but is not sufficient for transposition ability. Rather, the relative amounts in which the differenttransposaseproducts necessary for transposition are produced seems to be critical and influenced by host factors. In addition, transposition efficiency might be further reduced by the lack of essentialcis-required sequences in the modified I/dSpm version used in this study, although similar constructs were successfully employed in Arabidopsis. Eventually, cross-talk with related endogenous transposable elements may affect the mobility of the maize transposon in rice. Indeed, an interaction of the maizetransposaseswith a rice En/Spm -homologous element was revealed, resulting in the specific suppression of an alternative transcript in the latter (Chapter 8). This finding demonstrates that interference is possible and trans-activation potentially could occur among elements belonging to the same transposon family in different species.Based on the results of these analyses, a core collection of knockout and gene detection Ac/Ds lines with active transposition could be selected as a basis for developing populations for (forward and) reverse genetic screenings. The propagation of lines containing multiple transposons and the preferential insertion into gene-rich regions will help reduce the number of plants that would have to be produced in order to saturate the genome with insertions. At present, over 10,000 stabilised T 2Ac/Ds transposon lines are being analyzed in 5 EU laboratories for transposon flanking sequences that by comparison to the complete and annotated rice sequences will reveal tagged genes of interest that can be used for reverse genetics.
Classical mutagenesis in higher plants
Koornneef, M. - \ 2002
In: Molecular Plant Biology / Gilmartin, P.M., Bowler, C., Oxford, GB : Oxford University Press - p. 1 - 10.
genetica - mutagenese - mutanten - plantenveredeling - mutagenen - genetics - mutagenesis - mutants - plant breeding - mutagens
For a long time, mutagenesis research in plants focused on crop improvement and, especially for crop plants, opimised protocols were developed with barley being one of the favourite species. However, the interest in mutagenesis has shifted to basic plant research in the last 20 years, when the power of mutant approaches in combination with molecular techniques to investigate the molecular nature of the genes became fully appreciated
Identification of potato genes involved in Phytophthora infestans resistance by transposon mutagenesis
Enckevort, L.J.G. van - \ 2000
Agricultural University. Promotor(en): E. Jacobsen; A. Pereira. - S.l. : S.n. - ISBN 9789058083432 - 144
aardappelen - solanum tuberosum - phytophthora infestans - plantenziekteverwekkende schimmels - ziekteresistentie - transposons - merken van genen - selectie - plantenveredeling - transpositie - mutagenese - potatoes - solanum tuberosum - phytophthora infestans - plant pathogenic fungi - disease resistance - transposable elements - transposition - mutagenesis - gene tagging - selection - plant breeding
The late blight disease, caused by the oomycete Phytophthora infestans (Mont.) de Bary, is a serious threat to the potato crop every growing season. This has, for example, led to the disastrous Irish famine in the middle of the 19 th century, and continued in the 20 th century to remain a serious problem for potato growers. Since the early 1980s P. infestans populations changed more rapidly and epidemics even increased in their severity. Resistance breeding stimulated the introduction of resistance genes ( R genes) from wild Solanum species into cultivated potato, Solanum tuberosum , but newly occurring virulent races of P. infestans circumvented these R gene mediated resistances and no cultivars with durable resistance were obtained. At the moment, methods using fungicides supervised by spraying control via decision support systems are the only available control measures.
Characteristic for R gene type mediated resistance reactions is the hypersensitive response (HR) leading to local cell death causing necrotic spots at the site of attempted infection. Genetic analysis of HR mediated resistances showed that activation of HR is highly specific and induced upon recognition between a specific R gene in the plant and a corresponding avirulence gene ( Avr gene) in the pathogen. Insights in the molecular mechanisms underlying this HR resistance reaction in Solanum species might facilitate the development of potato cultivars that are more durable in maintaining a resistance phenotype.
A two component Ac-Ds transposon tagging strategy in diploidised potato was developed to identify and isolate genes involved in the R1 gene mediated resistance response to P. infestans . Transposable elements are molecular genetic tools to mutate and identify genes. The transposable elements Ac and Ds were first characterised in maize and their molecular isolation led to the identification of maize genes that were tagged by these elements. The autonomous Ac element is able to transpose by itself and also to induce transposition of the non-autonomous Ds element that is transposase defective. Introduction of these elements in heterologous species demonstrated their utility for isolating genes in self-fertilising plant species. Also in the highly heterozygous and tetraploid potato, the Ac and Ds transposable elements were shown to be functional. A cell autonomous visual marker gene for potato, the granule bound starch synthase gene ( GBSS gene), enabled a refined characterisation of Ac transposition in potato. Further molecular characterisation showed high levels of Ac-Ds transposition both somatically and germinally, so that suitable populations could be generated for tagging purposes.
The production of clones homozygous for the gene of interest that are normally required for efficient tagging strategies, turned out to be time consuming in potato due to self-incompatibility at the diploid level. Therefore, an alternative method based on somatic transposition was developed for the direct selection of transposition events instead of recovering germinally transmitted transpositions. Highly chimaeric Ac-Ds seedlings with active Ds transposition linked to the R1 resistance gene on chromosome 5 of potato were selected. Protoplasts were isolated from actively transposing seedlings and using the hygromycin excision selection marker, regenerants could be selected with new independent Ds insertions. The resulting R1 resistant transposon mutagenised population of almost 2000 hygromycin resistant regenerants formed an ideal start for the identification of an R1 tagged mutant, or other Ds insertional mutants with an altered R1 resistance response.
The somatically regenerated tagging population was analysed for the P. infestansR1 type HR resistance response, using a detached leaf assay for P.infestans inoculation. In a primary screening, 33 potential R1 resistance variants showing partial susceptibility to P. infestans race 0 were identified. These results were further quantified using stringent inoculation conditions on replicate samples leading to the identification of four putative mutants with a distinctly altered R1 resistance response. In these putative mutants less than 50% of the inoculated leaves showed the R1 type HR response and clear colonisation with sporulation of P. infestans was observed. The flanking sequences of the Ds insertion sites in these putative R1 mutants were analysed and in two cases a potential biological correlation between the insertion sequences and the phenotype was evident. One putative mutant contained a Ds insertion in a region with auxin and abscisic acid response cis-elements homologous to a specific region (TAPIR) of the tomato defence related genes TAPG2 and TAP1 .
The second P. infestansR1 resistance mutant, mutant 1000 with a striking susceptible phenotype was characterised in more detail. Two Ds insertions were identified and the insertion site flanking sequences both showed high homology to serine/threonine protein kinases. The Ds insertion sites turned out to be homologous but not identical, indicating two independent Ds insertions in homologous but not identical genes. Both sequences showed protein identity to all the conserved regions of serine/threonine protein kinases and they contained a conserved intron position. The closest homology was to the serine/threonine protein kinase domain of the Xanthomonas resistance gene Xa21 , which is involved in the induction of a HR resistance response in rice. This indicates that the isolated Solanum tuberosum protein kinase (StPK) homologs are candidate genes involved in resistance gene activity in potato. Further specific molecular analyses identified at least 11 homologs by sequence, which probably belong to a large family of serine/threonine protein kinases in potato. Both homologs in which the Ds transposons are inserted were present in susceptible parental potato clones. Therefore, it is unlikely that the isolated sequences represent the R1 gene itself. The mutated StPKs were designated rpr1 and rpr2 , r equired for P hytophthora infestansr esistance gene 1 and 2. Studying these mutants and the StPKs involved might help in understanding the pathway leading to HR resistance in potato.
Coenzyme recognition in para-hydroxybenzoate hydroxylase
Eppink, M.H.M. - \ 1999
Agricultural University. Promotor(en): N.C.M. Laane; W.J.H. van Berkel; H.A. Schreuder. - S.l. : Eppink - ISBN 9789058080653 - 167
oxygenasen - benzoaten - mutagenese - co-enzymen - oxygenases - benzoates - mutagenesis - coenzymes
Biochemistry is the science that studies the chemistry of life. This 'biological' chemistry includes growth, differentiation, movement, conductivity, immunity, transport and storage. During these processes proteins play an important role. The building blocks of proteins are amino acids, of which twenty are known. With these building blocks at hand it is possible to construct numerous proteins with many specific functions. A protein is not an elongated chain of amino acid residues but a compact very well defined three-dimensional structure. Two basic substructures are known in a protein, a cylindricalα-helix and an elongatedβ-strand. A number of theseα-helices and/orβ-strands connected by loop regions form a protein domain and a protein is built up of one or more domains. Furthermore, proteins can contain certain motifs (folds), structural conserved patterns. A large group of proteins with similar function and/or structure are called a protein family.
A special group of proteins, called enzymes or biocatalysts, are able to increase the rate of a chemical reaction by lowering the activation energy of that reaction. Enzymes are highly specific, because they influence the reactivity of the substrate in such a way that the substrate is quickly and efficiently converted into a product. Moreover, flexible/dynamic movements in enzymes may play an important role during catalysis, because enzymes are not always rigid bodies. To control the reaction, enzymes often need cofactors. Some examples are the already mentioned dinucleotides NAD(P)H and FAD, that play a role in electron transfer (redox) reactions. Generally speaking, these cofactors bind very specific to a protein. A well-known binding motif for NAD(P)H and FAD in different enzyme families is the Rossmann fold (Chapter 1), discovered by Michael Rossmann in 1974.
The NAD(P)H cofactor binds to the enzyme, electron transfer takes place and finally, the oxidized cofactor is released. In some proteins, the mode of NADPH binding is unknown. One example is p -hydroxybenzoate hydroxylase (PHBH), a flavoprotein monooxygenase that belongs to the family of FAD-dependent aromatic hydroxylases.
FAD-dependent Aromatic Hydroxylases
FAD-dependent aromatic hydroxylases play a role in the biodegradation of aromatic compounds. In nature, these compounds occur in plant polymers (lignin) as well as in proteins, steroïds and terpenes. During this century, the natural pool of aromatic compounds has been extended with products of industrial origin. Many of these synthetic compounds (pesticides, herbicides, fungicides and detergents) place a heavy burden on the environment and accumulate in soil and sludge. Microbial FAD-dependent aromatic hydroxylases catalyze the conversion of natural and synthetic aromatic substrates into products that can be further degraded to carbon dioxide and water. Recently, it was found that these enzymes are also involved in the biosynthesis of steroïds, plant hormones and antibiotics. PHBH is the archetype (prototype) of the family of FAD-dependent aromatic hydroxylases. In Wageningen, research on PHBH and related enzymes is embedded in the Wageningen Graduate School of Environmental Chemistry & Toxicology.
p -Hydroxybenzoate Hydroxylase
p -Hydroxybenzoate hydroxylase is isolated from the soil bacterium Pseudomonas fluorescens . This microbe can grow on 4-hydroxybenzoate (POHB) and other aromatic compounds as sole carbon source. PHBH catalyzes the conversion of POHB into 3,4-dihydroxybenzoate (DOHB) in the presence of NADPH and molecular oxygen. DOHB is a common intermediate in the aerobic degradation of plant material. After ring cleavage of DOHB and further degradation, the final products acetyl coenzyme A and succinate are fed into the citric acid cycle to provide energy for the cell.
p -Hydroxybenzoate hydroxylase has been subject to detailed kinetic and structural studies. The three-dimensional structure of PHBH is built up of three domains (Chapter 1). The first domain is the FAD-binding domain with the specific Rossmann fold for binding the ADP part of FAD. The second domain is the substrate-binding domain and the third domain (interface domain) is important for the interaction with another PHBH subunit, because PHBH exists as a dimer.
The structure of the enzyme-substrate complex is known in atomic detail. Recently, it was found that the flavin ring is able to move between an "open" and "closed" conformation. This flavin mobility is important for substrate binding and product release. However, unknown is the NADPH-binding site and where the reaction between NADPH and FAD takes place. Related questions are:
-Which amino acids play a role in cofactor binding?
-Is there a particular sequence motif for cofactor binding?
Another very important question concerns the effector role of the substrate. Upon binding of the aromatic substrate the flow of electrons from NADPH to FAD is 105 times enhanced. However, the molecular principles of this control are poorly understood. In this thesis we have tried to shed more light on the coenzyme recognition by PHBH.
Flavin ring mobility
In Chapter 2 the FAD in PHBH is substituted by a modified FAD, normally present in alcohol oxidase from methylotrophic yeasts. The crystal structure of p -hydroxybenzoate hydroxylase with this flavin analog not only represents the first crystal structure of an enzyme reconstituted with a modified flavin, but also provides direct evidence for the presence of an arabityl sugar chain in the modified form of FAD. The reconstituted enzyme-substrate complex shows that the flavin ring attains the "open" conformation. In the native enzyme-substrate complex the flavin ring is located in the "closed" conformation. The rate of flavin reduction by NADPH is much more rapid as compared to the native enzyme-substrate complex, suggesting that the mobility of the flavin ring is essential for the efficient reduction of the enzyme/substrate complex.
Amino acids involved in NADPH binding
To investigate the mode of NADPH binding, several amino acid residues were replaced by site-directed mutagenesis. The amino acids were selected on the basis of earlier results from chemical modification, crystallographic and modeling studies. Chapters 3, 4, 6 and 8 describe the properties of single mutants. It is concluded that Arg33, Gln34, Tyr38, Arg42, Arg44, His162 and Arg269 are involved in NADPH binding.
Structural motif for NADPH binding
PHBH contains two conserved sequence motifs, both involved in FAD binding. Chapter 5 describes a new unique sequence motif for the family of FAD-dependent aromatic hydroxylases, putatively involved in both FAD and NAD(P)H binding. From the recently determined crystal structure of phenol hydroxylase it is deduced that this sequence motif is also structurally conserved. Chapter 6 and 7 show that only His162 of this novel motif is directly important for the binding of NADPH.
Chapter 8 describes the cloning, purification and characterization of PHBH from Pseudomonas species CBS3. This is the first PHBH enzyme with known sequence that is active with NADH. Based on sequence analysis and homology modelling it is proposed that the helix H2 region is important for the binding of the 2'-phosphate moiety of NADPH. In Chapter 9 , the coenzyme specificity of PHBH from Pseudomonas fluorescens was addressed in further detail. Multiple replacements in helix H2 showed that Arg33 and Tyr38 are crucially involved in determining the coenzyme specificity. For the first time, a PHBH enzyme was constructed, which is more efficient with NADH.
Substrate binding is essential for a rapid reduction of FAD. This allows the subsequent attack of oxygen and the formation of the flavinhydroperoxide hydroxylating species. The question arises whether the stimulating effect of substrate binding on flavin reduction is caused by a large conformational change or merely due to subtle rearrangements in the active site. Chapter 10 describes the crystal structure of the substrate-free enzyme. This study shows that no large conformational changes take place upon substrate (analog) binding. The stimulating role of POHB is probably caused by several subtle effects. Stabilisation of the phenolate form of the substrate results in distribution of the electronic charges in the active site. These charge distributions influence the dynamic equilibrium between the "open" and "closed" conformation of FAD in such a way that the nicotinamide ring of NADPH and the isoalloxazine ring of FAD become optimally oriented for efficient reduction.