By first coming to understand the properties of R-HPCDH through structural analysis, we subsequently 1) constructed a pair wise sequence alignment between a R- and S-HPCDH to discover how they may differ and then 2) built structural models based on the sequence alignment to determine the basis of stereoselectivity in this enzyme family. Specifically, we discovered that the two positively charged residues (Arg152 and Arg196) that bind the sulfonate moiety of R-HPC are not conserved between R and S-HPCDH. Thus the sulfonate binding site is located in different places depending on whether the HPCDH selectively catalyzes R- or S-HPC. The sequence alignment alone fails to identify which positively charged residues might serve this function in S-HPCDH because it does not give us information on the location of residues in 3D space.
We discovered that the structural model supports Arg203 and Lys206 as the residues that bind the sulfonate moiety of S-HPC. Curiously, the sequence alignment in this region is as follows:
Thus, a positively charged residue exists at position 206 (S-HPCDH numbering) in both HPCDHs.
Based on this could we predict which HPCDH has a greater substrate selectivity (a greater differential in enzyme activity between R- and S-HPC)?
A theory now exists with some experimental support that enzymes with specific activities (they bind a single specific substrate and produce a single specific product) evolved from enzymes that were more promiscuous (enzymes that acted on multiple substrates and produced multiple products).
How might we engineer R-HPCDH to be more specific towards catalyzing the oxidation of R-HPC over S-HPC?
Finally, this tutorial is based on published research.
Clark et al. The stereoselectivity and catalytic properties of Xanthobacter autotrophicus 2-[(R)-2-Hydroxypropylthio]ethanesulfonate dehydrogenase are controlled by interactions between C-terminal arginine residues and the sulfonate of coenzyme M. Biochemistry (2004) vol. 43 (21) pp. 6763-71
Students likely followed some of the very steps that the authors took to come to the same conclusion. Congratulations on completion of the NRBSC Comparative Modeling Tutorial!