Department of Chemistry, University of Georgia, Athens, Georgia 30602, United States.
Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602, United States.
Biochemistry. 2021 May 25;60(20):1609-1618. doi: 10.1021/acs.biochem.1c00106. Epub 2021 May 5.
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 serovar typhimurium. We have determined the crystal structure of DGL by SAD phasing with selenomethionine to a resolution of 2.58 Å. 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 α-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 DO 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 - 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.
d-葡糖胺-6-磷酸氨裂解酶（DGL）是一种依赖于吡哆醛 5'-磷酸（PLP）的酶，可在鼠伤寒沙门氏菌中代谢 d-葡糖胺时产生 2-酮-3-脱氧葡糖酸 6-磷酸（KDG-6-P）。我们通过使用硒代蛋氨酸的 SAD 相位测定法将 DGL 的晶体结构解析至 2.58 Å 的分辨率。该序列与大多数其他氨基转移酶（AT）超家族成员的序列同一性非常低。结构形成八聚体组装，作为二聚体的四聚体，这在 AT 超家族中以前没有观察到。PLP 以希夫碱的形式共价结合到催化二聚体中位于两个单体界面处的 Lys-213。该结构缺乏在 AT 超家族的大多数成员中结合底物α-羧酸盐的保守精氨酸。然而，在小结构域中存在一个精氨酸簇，可能作为底物磷酸盐的结合位点。在 DO 中进行的脱氨反应使 KDG-6-P 产物特异性地在 C3 处氘化；因此，该机制必须涉及烯胺中间体，该中间体在产物释放前被酶质子化。核磁共振（NMR）分析表明，氘位于产物的-位置，表明在 C3 处构型反转的情况下发生水的消除，这对于依赖于 PLP 的脱水酶/脱氨酶来说是前所未有的。基于晶体结构和 NMR 数据，提出了 DGL 的反应机制。
