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,Biochemistry

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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
Biochemistry ( IF 2.9 ) Pub Date : 2021-05-05 , DOI: 10.1021/acs.biochem.1c00106
Robert S Phillips _{1,

2} , Samuel C-K Ting ₂ , Kaitlin Anderson ₃

Affiliation

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 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 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.

中文翻译：

d-葡萄糖胺-6-磷酸氨裂解酶的结构和机制：吡哆醛 5'-磷酸依赖性酶的新型八聚体组装，以及 d-氨基酸消除反应中前所未有的立体化学反转

d -葡萄糖胺-6-磷酸氨裂解酶 (DGL) 是一种吡哆醛 5′-磷酸 (PLP) 依赖性酶，在d代谢中产生 2-酮-3-脱氧葡萄糖酸 6-磷酸 (KDG-6-P) -由鼠伤寒沙门氏菌产生的葡萄糖胺酸。我们通过硒代蛋氨酸 SAD 定相确定了 DGL 的晶体结构，分辨率为 2.58 Å。该序列与转氨酶 (AT) 超家族的大多数其他成员的同一性非常低。该结构形成八聚体组装体，作为二聚体的四聚体，此前在 AT 超家族中尚未观察到。 PLP 作为席夫碱与两个单体界面处催化二聚体中的 Lys-213 共价结合。在 AT 超家族的大多数成员中，该结构缺乏与底物 α-羧酸盐结合的保守精氨酸。然而，在小结构域中存在一簇精氨酸，可能充当底物磷酸盐的结合位点。在D ₂ O中进行脱氨反应，得到C3立体定向氘化的KDG-6-P产物；因此，该机制必须涉及在产品释放之前被酶质子化的烯胺中间体。核磁共振（NMR）分析表明，氘位于产物中的pro - R位置，这表明水的消除是随着C3构型的反转而发生的，这对于PLP依赖性脱水酶/脱氨酶来说是前所未有的。根据晶体结构和 NMR 数据，提出了 DGL 的反应机理。

更新日期：2021-05-25

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