Background

We discovered in Step 4 through a pairwise sequence alignment between R- and S-HPCDH that 1) the residues that bind the hydroxyl moiety of R-HPC and assist in catalyzing the reaction are conserved and 2) that the most dramatic change is that the positively charged residues (two Arginines) that bind the sulfonate moiety of R-HPC are not conserved. Thus, it seems logical to hypothesize that the sulfonate and methyl binding sites have exchanged positions in the enzyme. S-HPC would then be expected to bind to S-HPCDH with the hydride directed toward NAD in contrast to how it binds in R-HPCDH (recall Step 3).

Nevertheless, we are still left wondering which specific positively charged residues are responsible for the basis of stereospecificity. This knowledge is useful to understanding the evolution/re-engineering of proteins and for a deeper knowledge of enzyme catalysis. There are 15 Arginines and 4 Lysines in S-HPCDH. An examination of all the Arginines (Arg) and Lysines (Lys) might be expensive both in money and time. The sequence alignment, though useful in several other contexts, fails to give us an answer as to which residues cause the switch in stereospecificity. Therefore, in Step 5 we constructed a comparative model of S-HPCDH using R-HPCDH as a template. The structural model can in many circumstances help narrow the scope of investigation by identifying positively charged residues that are positioned to properly bind the sulfonate group of S-HPC.

In this final step, we will visualize the structure and identify the residues that determine the basis of stereospecificity.