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Distant Non-Obvious Mutations Influence the Activity of a Hyperthermophilic Pyrococcusfuriosus Phosphoglucose Isomerase
Subramanian, Kalyanasundaram ; Mitusińska, Karolina ; Raedts, John ; Almourfi, Feras ; Joosten, Henk Jan ; Hendriks, Sjon ; Sedelnikova, Svetlana E. ; Kengen, Servé W.M. ; Hagen, Wilfred R. ; Góra, Artur ; Martins Dos Santos, Vitor A.P. ; Baker, Patrick J. ; Oost, John van der; Schaap, Peter J. - \ 2019
Biomolecules 9 (2019)6. - ISSN 2218-273X
Comulator - cupin phosphoglucose isomerase - Protein engineering - Pyrococcus furiosus - solvent access
The cupin-type phosphoglucose isomerase (PfPGI) from the hyperthermophilic archaeon Pyrococcus furiosus catalyzes the reversible isomerization of glucose-6-phosphate to fructose-6-phosphate. We investigated PfPGI using protein-engineering bioinformatics tools to select functionally-important residues based on correlated mutation analyses. A pair of amino acids in the periphery of PfPGI was found to be the dominant co-evolving mutation. The position of these selected residues was found to be non-obvious to conventional protein engineering methods. We designed a small smart library of variants by substituting the co-evolved pair and screened their biochemical activity, which revealed their functional relevance. Four mutants were further selected from the library for purification, measurement of their specific activity, crystal structure determination, and metal cofactor coordination analysis. Though the mutant structures and metal cofactor coordination were strikingly similar, variations in their activity correlated with their fine-tuned dynamics and solvent access regulation. Alternative, small smart libraries for enzyme optimization are suggested by our approach, which is able to identify non-obvious yet beneficial mutations.
Genetic monitoring to evaluate reintroduction attempts of a highly endangered rodent
Haye, M.J.J. la; Reiners, T.E. ; Raedts, R. ; Verbist, V. ; Koelewijn, H.P. - \ 2017
Conservation Genetics 18 (2017)4. - ISSN 1566-0621 - p. 877 - 892.
Admixture - Captive breeding - Common hamster - Effective and census population size - Founder effect - Genetic variation
The ultimate strategy to prevent species extinction is captive breeding followed by reintroduction of individuals into the wild. Unfortunately, overall success of reintroductions is poor and in most cases conservation breeding is applied for species where individual numbers, population numbers and genetic diversity is strongly reduced. In addition, reintroductions inevitably result in small populations with poor genetic status. Systematic demographic and genetic monitoring is needed to optimize conservation actions. Here we show how genetic monitoring was useful and informative in a reintroduction project for the highly endangered Common hamster (Cricetus cricetus) in the Netherlands and Belgium. Using well defined breeding lines of original relict populations combined with a systematic reintroduction scheme including consecutive supplementations made it possible to infer success of reintroduction, supplementation and following admixture of populations. An initial loss of genetic diversity could be detected in some of the reintroduced populations, but it could be shown that due to following supplementation of populations, genetic diversity and also effective population size in the wild stabilized or even increased. Multivariate (DAPC) and Bayesian inference (STRUCTURE) revealed admixture of supplemented individuals with wild-born individuals. Although population size estimates differed strongly between populations, a link between census size, breeding lines, effective population size and genetic diversity could not be proven. This study highlights that genetic monitoring is not only descriptive but also reveals detailed information on reintroduction success, admixture and population development. We recommend that genetic monitoring should be a basic element of reintroductions and should be used to optimize reintroduction attempts.
Molecular Characterization of an NADPH-Dependent Acetoin Reductase/2,3-Butanediol Dehydrogenase from Clostridium beijerinckii NCIMB 8052
Raedts, J.G.J. ; Siemerink, M.A.J. ; Levisson, M. ; Oost, J. van der; Kengen, S.W.M. - \ 2014
Applied and Environmental Microbiology 80 (2014)6. - ISSN 0099-2240 - p. 2011 - 2020.
l-threonine dehydrogenase - protein-structure prediction - crystal-structure - alcohol dehydrogenases - (2r,3r)-2,3-butanediol dehydrogenase - thermoanaerobacter-brockii - escherichia-coli - meso-2,3-butanediol dehydrogenase - sulfolobus-solfataricus - butanol fer
Acetoin reductase is an important enzyme for the fermentative production of 2,3-butanediol, a chemical compound with a very broad industrial use. Here, we report on the discovery and characterization of an acetoin reductase from Clostridium beijerinckii NCIMB 8052. An in silico screen of the C. beijerinckii genome revealed eight potential acetoin reductases. One of them (CBEI_1464) showed substantial acetoin reductase activity after expression in Escherichia coli. The purified enzyme (C. beijerinckii acetoin reductase [Cb-ACR]) was found to exist predominantly as a homodimer. In addition to acetoin (or 2,3-butanediol), other secondary alcohols and corresponding ketones were converted as well, provided that another electronegative group was attached to the adjacent C-3 carbon. Optimal activity was at pH 6.5 (reduction) and 9.5 (oxidation) and around 68°C. Cb-ACR accepts both NADH and NADPH as electron donors; however, unlike closely related enzymes, NADPH is preferred (Km, 32 µM). Cb-ACR was compared to characterized close homologs, all belonging to the "threonine dehydrogenase and related Zn-dependent dehydrogenases" (COG1063). Metal analysis confirmed the presence of 2 Zn(2+) atoms. To gain insight into the substrate and cofactor specificity, a structural model was constructed. The catalytic zinc atom is likely coordinated by Cys37, His70, and Glu71, while the structural zinc site is probably composed of Cys100, Cys103, Cys106, and Cys114. Residues determining NADP specificity were predicted as well. The physiological role of Cb-ACR in C. beijerinckii is discussed
A novel bacterial enzyme with D-glucuronyl C5-epimerase activity
Raedts, J.G.J. ; Lundgren, M. ; Kengen, S.W.M. ; Li, J.P. ; Oost, J. van der - \ 2013
Journal of Biological Chemistry 288 (2013)34. - ISSN 0021-9258 - p. 24332 - 24339.
l-iduronic acid - heparan-sulfate - heparin/heparan sulfate - escherichia-coli - biosynthesis - polysaccharide - glycosaminoglycans - oligosaccharides - purification - database
Glycosaminoglycans are biologically active polysaccharides that are found ubiquitously in the animal kingdom. The biosynthesis of these complex polysaccharides involves complicated reactions that turn the simple glycosaminoglycan backbone into highly heterogeneous structures. One of the modification reactions is the epimerization of D-glucuronic acid to its C5-epimer L-iduronic acid, which is essential for the function of heparan sulfate. Although L-iduronic acid residues have been shown to exist in polysaccharides of some prokaryotes, there has been no experimental evidence for the existence of a prokaryotic D-glucuronyl C5-epimerase. This work for the first time reports on the identification of a bacterial enzyme with D-glucuronyl C5-epimerase activity. A gene of the marine bacterium Bermanella marisrubri sp. RED65 encodes a protein (RED65_08024) of 448 amino acids that has an overall 37% homology to the human D-glucuronic acid C5-epimerase. Alignment of this peptide with the human and mouse sequences revealed a 60% similarity at the carboxyl terminus. The recombinant protein expressed in Escherichia coli showed epimerization activity toward substrates generated from heparin and the E. coli K5 capsular polysaccharide, thereby providing the first evidence for bacterial D-glucuronyl C5-epimerase activity. These findings may eventually be used for modification of mammalian glycosaminoglycans
|Genetic diversity in reintroduced and restocked populations of the Common hamster (Cricetus cricetus)
Raedts, R. ; Kats, R.J.M. van; Koelewijn, H.P. ; Kuiters, A.T. ; Muskens, G.J.D.M. ; Haye, M.J.J. la - \ 2011
In: Proceedings of the 16t and 17th Meeting of the International Hamster Workgroup; Ranis, Germany (2009), Gödollo, Hungary (2010). - p. 107 - 116.
Molecular characterization and optimization of enzymes involved in glycosaminoglycan biosynthesis
Raedts, J.G.J. - \ 2011
Wageningen University. Promotor(en): John van der Oost, co-promotor(en): Servé Kengen. - [S.l.] : S.n. - ISBN 9789461730190 - 154
glycosaminoglycanen - hyaluronzuur - heparine - biosynthese - escherichia coli - genexpressie - glycosaminoglycans - hyaluronic acid - heparin - biosynthesis - escherichia coli - gene expression
Glycosaminoglycans are biological active polysaccharides composed of repeating disaccharides composed of a hexuronic acid and a hexosamine. They have various pharmaceutical applications and traditionally this type of molecule is isolated from animal tissue. Since extraction from animal derivatives has serious limitations for the production of a large variety of defined glycosaminoglycans, there is a general interest in developing alternative systems enabling a more tightly controlled synthesis. During this research project we explored alternative ways of controlled chemo-enzymatic synthesis ofmonodisperse and uniform glycosaminoglycans for pharmaceutical applications, with a main focus on heparin. Heparin is a highly sulfated and complex glycosaminoglycan which is worldwide used as an anticoagulant to prevent blood clotting during surgery.Upon closer investigation of the heparin biosynthesis the D-glucuronyl C5-epimerase was recognized as a key enzyme, as this is the only reaction that cannot be done chemically. Two alternative approaches were taken to get closer to an industrial applicable enzyme; improvement of the animal heparin sulfate D-glucuronyl C5-epimerase and identification and isolation of novel candidate C5-epimerases from prokaryotes. Both approaches gave functional production of C5-epimerases.
Occurrence of L-iduronic acid and putative D-glucuronyl C5-epimerases in prokaryotes
Raedts, J.G.J. ; Kengen, S.W.M. ; Oost, J. van der - \ 2011
Glycoconjugate Journal 28 (2011)2. - ISSN 0282-0080 - p. 57 - 66.
multiple sequence alignment - k5 capsular polysaccharide - escherichia-coli - pasteurella-multocida - o-antigens - identification - biosynthesis - heparin - constituent - lipopolysaccharide
Glycosaminoglycans (GAGs) are polysaccharides that are typically present in a wide diversity of animal tissue. Most common GAGs are well-characterized and pharmaceutical applications exist for many of these compounds, e.g. heparin and hyaluronan. In addition, also bacterial glycosaminoglycan-like structures exist. Some of these bacterial GAGs have been characterized, but until now no bacterial GAG has been found that possesses the modifications that are characteristic for many of the animal GAGs such as sulfation and C5-epimerization. Nevertheless, the latter conversion may also occur in bacterial and archaeal GAGs, as some prokaryotic polysaccharides have been demonstrated to contain L-iduronic acid. However, experimental evidence for the enzymatic synthesis of L-iduronic acid in prokaryotes is as yet lacking. We therefore performed an in silico screen for D-glucuronyl C5-epimerases in prokaryotes. Multiple candidate C5-epimerases were found, suggesting that many more microorganisms are likely to exist possessing an L-iduronic acid residue as constituent of their cell wall polysaccharides
Cofactor engineering of Lactobacillus brevis alcohol dehydrogenase by computational design
Machielsen, M.P. ; Looger, L.L. ; Raedts, J.G.J. ; Dijkhuizen, S. ; Hummel, W. ; Henneman, H.G. ; Daussmann, T. ; Oost, J. van der - \ 2009
Engineering in Life Sciences 9 (2009)1. - ISSN 1618-0240 - p. 38 - 44.
escherichia-coli - proteins - reduction - evolution - enzyme
The R-specific alcohol dehydrogenase from Lactobacillus brevis (Lb-ADH) catalyzes the enantioselective reduction of prochiral ketones to the corresponding secondary alcohols. It is stable and has broad substrate specificity. These features make this enzyme an attractive candidate for biotechnological applications. A drawback is its preference for NADP(H) as a cofactor, which is more expensive and labile than NAD(H). Structure-based computational protein engineering was used to predict mutations to alter the cofactor specificity of Lb-ADH. Mutations were introduced into Lb-ADH and tested against the substrate acetophenone, with either NAD(H) or NADP(H) as cofactor. The mutant Arg38Pro showed fourfold increased activity with acetophenone and NAD(H) relative to the wild type. Both Arg38Pro and wild type exhibit a pH optimum of 5.5 with NAD(H) as cofactor, significantly more acidic than with NADP(H). These and related Lb-ADH mutants may prove useful for the green synthesis of pharmaceutical precursors
NSP1 of the GRAS protein family is essential for Rhizobial Nod factor-induced transcription
Smit, P. ; Raedts, J.G.J. ; Portyanko, V. ; Debellé, F. ; Gough, C. ; Bisseling, T. ; Geurts, R. - \ 2005
Science 308 (2005)5729. - ISSN 0036-8075 - p. 1789 - 1791.
receptor kinase gene - medicago-truncatula - bacterial - fungal - identification - transduction - symbiosis - mutants - calcium
Rhizobial Nod factors induce in their legume hosts the expression of many genes and set in motion developmental processes leading to root nodule formation. Here we report the identification of the Medicago GRAS-type protein Nodulation signaling pathway 1 (NSP1), which is essential for all known Nod factor–induced changes in gene expression. NSP1 is constitutively expressed, and so it acts as a primary transcriptional regulator mediating all known Nod factor–induced transcriptional responses, and therefore, we named it a Nod factor response factor.
|Isolation of proteins from potato-starch granules using a composite gel on the Model 230A micropreperative electrophoresis system.
Suurs, L.C.J.M. ; Raedts, M. - \ 1993
Biosystems Reporter 18 (1993). - p. 6 - 7.