Determinants of substrate specificity in D-3-phosphoglycerate dehydrogenase. Conversion of the M. tuberculosis enzyme from one that does not use α-ketoglutarate as a substrate to one that does.

ABSTRACT

d-3-Phosphoglycerate dehydrogenase (PGDH) converts d-3-phosphoglycerate (PGA) to phosphohydroxypyruvate (PHP) in the first step of l-serine biosynthesis. This reaction is reversible, and some PGDHs are capable of using α-ketoglutarate (αKG) instead of PHP in the reverse direction to produce α-hydroxyglutarate. The enzymes so far shown to have this ability are Type II PGDHs, suggesting that this may be a common feature of the Type II enzymes. Type I PGDHs examined so far do not share this feature. Inspection of PGDH sequences shows that a GCFCI … WXKX motif is commonly found in Type II PGDHs while a GRAGT … WXRX motif is commonly associated with Type I PGDHs. The removal of the cationic side chain at the first position shown above in the Type I PGDH from Mycobacterium tuberculosis converts it to an enzyme capable of using αKG where the native enzyme is not. It also produces an enzyme that regenerates NAD+ in the forward reaction when coupled to phosphoserine aminotransferase, as was previously shown for E. coli PGDH. Substitution of an arginyl residue for a lysyl residue at the second position of ecPGDH, decreases the kcat/Km of the enzyme by approximately 50-fold when using αKG, but only approximately 3-fold when using PHP. This suggests that a PGDH dependent cycle that conserves NAD+ in E. coli may be operative in many other organisms expressing the GCFCI … WXKX motif.