Disruption of active site interactions with pyridoxal 5'-phosphate and substrates by conservative replacements in the glycine-rich loop of Escherichia coli D-serine dehydratase

Abstract

We have used site-directed mutagenesis to examine the function of three putative active site residues (C278, G279, and G281) of the vitamin B6 enzyme D-serine dehydratase. These residues lie in or adjacent to a conserved glycine-rich loop that is known to interact with the pyridoxal 5'-phosphate cofactor in several B6 enzymes and that resembles the GXGXXG loop of nucleotide-binding sites. The cofactor affinity, catalytic properties, and spectral properties (UV, CD, fluorescence, and 31P NMR) of alanine variants C278A, G279A, and G281A were measured as well as the susceptibility of each variant to thiol modification by 5,5'-dithiobis(2-nitrobenzoic acid). The specific thiols modified in each variant and wild type D-serine dehydratase were identified by amino acid sequencing of labeled tryptic peptides. C278A, G279A, and G281A displayed 10-, 33-, and 22-fold lower affinities for pyridoxal 5'-phosphate than did wild type D-serine dehydratase and turnover numbers with D-serine that were 50, 6, and 60% of normal, respectively. The introduction of a methyl side chain into G281 enhanced catalytic efficiency with the substrates D-threonine, D-allo-threonine, and L-serine, whereas the methyl side chain at position 279 impaired catalysis of all substrates as well as cofactor affinity. The 31P NMR spectrum of D-serine dehydratase was minimally perturbed by the alanine substitutions, consistent with the view that neither G279 nor G281 interacts with the phosphate group of the cofactor (in contrast to the arrangement found in several other B6 enzymes). C311 was the single thiol modified by 5,5'-dithiobis(2-nitrobenzoic acid) in wild type D-serine dehydratase. Two normally inaccessible thiol groups, C233 and C278, were rendered susceptible to modification as a consequence of either G----A substitution, and modification of C278 was associated with inactivation of G279A and G281A. These observations suggest that small perturbations in the glycine-rich loop induce conformational changes spanning a considerable area around the active site.