Properties and mechanism of d-glucosaminate-6-phosphate ammonia-lyase: An aminotransferase family enzyme with d-amino acid specificity.

Salmonella enterica serovar Typhimurium utilizes a wide range of growth substrates, some of which are relatively novel. One of these unusual substrates is d-glucosaminate, which is metabolized by the enzymes encoded in the dga operon. d-Glucosaminate is transported and converted to d-glucosaminate-6-phosphate (G6P) by a phosphotransferase system, composed of DgaABCD. The protein product of dgaE, d-glucosaminate-6-phosphate ammonia lyase (DGL), converts G6P to 2-keto-3-deoxygluconate-6-phosphate, which undergoes a retroaldol reaction catalyzed by the DgaF protein to give d-glyceraldehyde-3-phosphate and pyruvate. We have now developed an improved synthesis of G6P which gives a higher yield. The DGL reaction is of mechanistic interest because it is one of only a few enzymes in the pyridoxal-5'-phosphate (PLP) dependent aminotransferase superfamily known to catalyze reaction of a d-amino acid substrate. The pH dependence of DGL shows an optimum at 7.5-8.5, suggesting a requirement for a catalytic base. α-Glycerophosphate and inorganic phosphate are weak competitive inhibitors, with Ki values near 30mM, and d-serine is neither a substrate nor an inhibitor. We have found in rapid-scanning stopped-flow experiments that DGL reacts rapidly with its substrate to form a quinonoid intermediate with λmax=480nm, within the dead time (ca. 2msec), which then rapidly decays (k=279s-1) to an intermediate with absorption between 330 and 350nm, probably an aminoacrylate complex. We suggest a mechanism for DGL and propose that the unusual stereochemistry of the DGL reaction requires a catalytic base poised on the opposite face of the PLP-substrate complex from the other members of the aminotransferase superfamily.

His-163 is a stereospecific proton donor in the mechanism of d-glucosaminate-6-phosphate ammonia-lyase.

d-Glucosaminate-6-phosphate ammonia-lyase (DGL) catalyzes the conversion of d-glucosaminate-6-phosphate to 2-keto-3-deoxyglutarate-6-phosphate, with stereospecific protonation of C-3 of the product. The crystal structure of DGL showed that His-163 could serve as the proton donor. H163A mutant DGL is fully active in the steady-state reaction, and the pre-steady-state kinetics are very similar to those of wild-type DGL. However, H163A DGL accumulates a transient intermediate with λmax at 293 nm during the reaction that is not seen with wild-type DGL. Furthermore, NMR analysis of the reaction of H163A DGL in D2 O shows that the product is a mixture of deuterated diastereomers at C-3. These results establish that His-163 is the proton donor in the reaction mechanism of DGL.