Serine hydroxymethyltransferase: mechanism of the racemization and transamination of D- and L-alanine

Abstract

The reaction specificity and stereochemical control of Escherichia coli serine hydroxymethyltransferase were investigated with D- and L-alanine as substrates. An active-site H228N mutant enzyme binds both D- and L-alanine with Kd values of 5 mM as compared to 30 and 10 mM, respectively, for the wild-type enzyme. Both wild-type and H228N enzymes form quinonoid complexes absorbing at 505 nm by catalyzing the loss of the alpha-proton from both D- and L-alanine. Racemization and transamination reactions were observed to occur with both alanine isomers as substrates. The relative rates of these reactions are quinonoid formation greater than alpha-proton solvent exchange greater than racemization greater than transamination. The observation that the rate of quinonoid formation with either alanine isomer is an order of magnitude faster than solvent exchange suggests that the alpha-protons from both D- and L-alanine are transferred to base(s) on the enzyme. The rate of racemization is 2 orders of magnitude slower than the formation of the quinonoid complexes. This latter difference in rate suggests that the quinonoid complexes formed from D- and L-alanine are not identical. The difference in structure of the two quinonoid complexes is proposed to be the active-site location of the alpha-protons lost from the two alanine isomers, rather than two orientations of the pyridoxal phosphate ring. The results are consistent with a two-base mechanism for racemization.

Upregulation of MetC Is Essential for d -Alanine-Independent Growth of an alr/dadX -Deficient Escherichia coli Strain

“…Serine hydroxymethyl transferase (GlyA) transaminates both L-alanine and D-alanine and also catalyzes an alanine racemase reaction (10,11,37). However, the reported GlyA racemase coactivity is not sufficient to fully compensate for the alr/dadX deletions in our study.…”

“…From the entire E. coli proteome, 56 proteins were predicted to belong to this category of enzymes using the TagIdent tool (18), of which several were found to be upregulated in BL21(DE3)⌬alr⌬dadX PR . Besides GlyA, which was previously reported to possess limited alanine racemase activity (10,11,37), MetB, MetC, Asd, TrpB, and YbdL were also annotated as PLP cofactor-requiring enzymes. Plasmids expressing these genes under the control of the T7 promoter were transformed into BL21(DE3)⌬alr⌬dadX.…”

Catalytic and Thermodynamic Properties of Tetrahydromethanopterin-dependent Serine Hydroxymethyltransferase from Methanococcus jannaschii

“…Addition of 200 mM of either L-or D-alanine to the enzyme resulted in a transamination reaction, as indicated by the decrease of absorbance at 424 nm and the concomitant formation of a new absorption band with a maximum at 324 nm. The 498-nm absorbing band, corresponding to a quinonoid intermediate, which is observed with the E. coli enzyme (16), is absent in the reaction catalyzed by mjSHMT. The kinetics of both reactions, which were carried out at 60°C, fitted well to the sum of two first-order processes.…”

Evolutionary relationships among pyridoxal‐5′‐phosphate‐dependent enzymes

“…kinetically minor reactions that correspond to the main reaction of another B, enzyme. Transamination has been found to occur as a side reaction in a-amino acid decarboxylases (Meister, 1990), racemization in aminotransferases (Kochhar and Christen, 1988), racemization and transamination in tryptophan synthase (Miles, 1987), and a-decarboxylation (Palekar et al, 1973), transamination and racemization (Shostak and Schirch, 1988) in glycine hydroxymethyltransferase. Certain synthases, e.g.…”