7lc0

Publication Abstract from PubMed

d-Glucosaminate-6-phosphate ammonia-lyase (DGL) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that produces 2-keto-3-deoxygluconate 6-phosphate (KDG-6-P) in the metabolism of d-glucosaminic acid by Salmonella enterica serovar typhimurium. We have determined the crystal structure of DGL by SAD phasing with selenomethionine to a resolution of 2.58 A. The sequence has very low identity with most other members of the aminotransferase (AT) superfamily. The structure forms an octameric assembly as a tetramer of dimers that has not been observed previously in the AT superfamily. PLP is covalently bound as a Schiff base to Lys-213 in the catalytic dimer at the interface of two monomers. The structure lacks the conserved arginine that binds the alpha-carboxylate of the substrate in most members of the AT superfamily. However, there is a cluster of arginines in the small domain that likely serves as a binding site for the phosphate of the substrate. The deamination reaction performed in D2O gives a KDG-6-P product stereospecifically deuterated at C3; thus, the mechanism must involve an enamine intermediate that is protonated by the enzyme before product release. Nuclear magnetic resonance (NMR) analysis demonstrates that the deuterium is located in the pro-R position in the product, showing that the elimination of water takes place with inversion of configuration at C3, which is unprecedented for a PLP-dependent dehydratase/deaminase. On the basis of the crystal structure and the NMR data, a reaction mechanism for DGL is proposed.

Structure and Mechanism of d-Glucosaminate-6-phosphate Ammonia-lyase: A Novel Octameric Assembly for a Pyridoxal 5'-Phosphate-Dependent Enzyme, and Unprecedented Stereochemical Inversion in the Elimination Reaction of a d-Amino Acid.,Phillips RS, Ting SC, Anderson K Biochemistry. 2021 May 25;60(20):1609-1618. doi: 10.1021/acs.biochem.1c00106., Epub 2021 May 5. PMID:33949189^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.