- Adrie H. Westphal (1)
- Willem J.H. Berkel van (2)
- J.J. Koehorst (1)
- Mieke M.E. Huijbers(older publications) (1)
- Mieke M.E. Huijbers (1)
Dimerization of Proline Dehydrogenase from Thermus thermophilus Is Crucial for Its Thermostability
Huijbers, Mieke M.E. ; Wu, Jenny W. ; Westphal, Adrie H. ; Berkel, Willem J.H. van - \ 2019
Biotechnology Journal 14 (2019)5. - ISSN 1860-6768
flavoprotein - protein oligomerization - thermostability - Thermus thermophilus - triosephosphate isomerase barrel
Thermus thermophilus proline dehydrogenase (TtProDH) catalyzes the first step in proline catabolism. The thermostable flavoenzyme consists of a distorted triosephosphate isomerase (TIM) barrel and three N-terminal helices: αA, αB, and αC. Using maltose-binding protein (MBP) fused constructs, it has been recently demonstrated that helix αC is crucial for TtProDH catalysis and for tetramerization through positioning of helix α8. Here, the structural features that determine the thermostability of TtProDH are reported. Selective disruption of two ion pairs in the dimerization interface of several MBP-TtProDH variants result in the formation of monomers. The newly created monomers have improved catalytic properties but their melting temperatures are decreased by more than 20 °C. Sequence comparison suggests that one of the ion-pairs involved in dimerization is unique for ProDHs from Thermus species. In summary, intermolecular ion-pairs improve the thermostability of TtProDH and a trade-off is made between thermostability and catalytic activity.
A more polar N-terminal helix releases MBP-tagged Thermus thermophilus proline dehydrogenase from tetramer-polymer self-association
Huijbers, Mieke M.E. ; Berkel, Willem J.H. van - \ 2016
Journal of Molecular Catalysis. B, Enzymatic 134 (2016). - ISSN 1381-1177 - p. 340 - 346.
Flavoprotein - Molecular self-association - Proline dehydrogenase - Protein oligomerization - Thermus thermophilus
Proline dehydrogenase (ProDH) is a ubiquitous flavoenzyme involved in the biosynthesis of . l-glutamate. ProDH is of interest for biocatalysis because the protein might be applied in multi-enzyme reactions for the synthesis of structurally complex molecules. We recently demonstrated that the thermotolerant ProDH from . Thermus thermophilus (TtProDH) is overproduced in . Escherichia coli when using maltose-binding protein (MBP) as a solubility tag. However, MBP-TtProDH and MBP-clipped TtProDH are prone to aggregation through non-native self-association. Here we provide evidence that the hydrophobic N-terminal helix of TtProDH is responsible for the self-association process. The more polar MBP-tagged F10E/L12E variant exclusively forms tetramers and exhibits excellent catalytic features over a wide range of temperatures. Understanding the hydrodynamic and catalytic properties of thermostable enzymes is important for the development of industrial biocatalysts as well as for pharmaceutical applications.
Effects of Argonaute on gene expression in Thermus thermophilus
Swarts, D.C. ; Koehorst, J.J. ; Westra, E.R. ; Schaap, P.J. ; Oost, J. van der - \ 2015
PRJEB8709 - ERP009732 - Thermus thermophilus
To investigate if TtAgo also has the potential to control RNA levels, we analyzed RNA-seq data derived from cultures of four T. thermophilus strain HB27 variants: wild type, TtAgo knockout (Δago), and either strain transformed with a plasmid. Additionally we determined the effect of TtAgo on expression of plasmid-encoded RNA and plasmid DNA levels. Background Eukaryotic Argonaute proteins mediate RNA-guided RNA interference, allowing both regulation of host gene expression and defense against invading mobile genetic elements. Recently, it has become evident that prokaryotic Argonaute homologs mediate DNA-guided DNA interference, and play a role in host defense. Argonaute of the bacterium Thermus thermophilus (TtAgo) targets invading plasmid DNA during and after transformation. Using small interfering DNA guides, TtAgo can cleave single and double stranded DNAs. Although TtAgo additionally has been demonstrated to cleave RNA targets complementary to its DNA guide in vitro, RNA targeting by TtAgo has not been demonstrated in vivo. Methods To investigate if TtAgo also has the potential to control RNA levels, we analyzed RNA-seq data derived from cultures of four T. thermophilus strain HB27 variants: wild type, TtAgo knockout (Δago), and either strain transformed with a plasmid. Additionally we determined the effect of TtAgo on expression of plasmid-encoded RNA and plasmid DNA levels. Results In the absence of exogenous DNA (plasmid), TtAgo presence or absence had no effect on gene expression levels. When plasmid DNA is present, TtAgo reduces plasmid DNA levels 4-fold, and a corresponding reduction of plasmid gene transcript levels was observed. We therefore conclude that TtAgo interferes with plasmid DNA, but not with plasmid RNA. Interestingly, TtAgo presence stimulates expression of specific endogenous genes, but only when exogenous plasmid DNA was present. Specifically, the presence of TtAgo directly or indirectly stimulates expression of CRISPR loci and associated genes, some of which are involved in CRISPR adaptation. This suggests that TtAgo-mediated interference with plasmid DNA stimulates CRISPR adaptation.