|
type |
Journal Article |
authors |
Phillips RS, Ting SC, Tetsadjio AG, Anderson KL, Friez KM, Miller KA, Hoover TR |
title |
Properties and mechanism of d-glucosaminate-6-phosphate ammonia-lyase: An aminotransferase family enzyme with d-amino acid specificity |
journal |
Biochim Biophys Acta |
Activity |
4.3.1.29 |
Family |
4.3.1.29 |
sel |
selected |
ui |
29277660 |
year |
(2018) |
volume |
1866 |
number |
7 |
pages |
799-805 |
| |
keywords |
Aminotransferase family; Pyridoxal-5′-phosphate; Reaction mechanism; d-Amino acid |
abstract |
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. |
last changed |
2018/10/05 11:53 |
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