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.
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.
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DOI：
10.1021/acs.biochem.1c00106
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发表时间：
2021-05-25
期刊：
影响因子：
2.9
通讯作者：
Anderson K
中科院分区：
文献类型：
--
作者：
Phillips RS;Ting SC;Anderson K
d -氨基葡萄糖-6-磷酸解氨酶（DGL）是一种吡哆醛5 ' -磷酸（PLP）依赖性酶，在肠沙门氏菌血清型鼠伤寒沙门氏菌代谢d -氨基葡萄糖酸的过程中产生2-酮-3-脱氧葡萄糖酸6-磷酸（KDG-6-P）。我们用硒代蛋氨酸的SAD相位测定了DGL的晶体结构，分辨率为2.58 Å。该序列与大多数其他转氨酶（AT）超家族成员的同源性很低。该结构形成了一个八聚体组装，作为二聚体的四聚体，这在以前的AT超家族中没有观察到。PLP作为希夫碱在催化二聚体的两个单体界面上与Lys-213共价结合。该结构在大多数AT超家族成员中缺乏与底物α-羧酸盐结合的保守精氨酸。然而，在小结构域中有一簇精氨酸，可能作为底物磷酸盐的结合位点。在D2O中进行脱氨反应，得到立体特异地在C3处氘化的KDG-6-P产物；因此，该机制必须涉及在产物释放前被酶质子化的烯胺中间体。核磁共振（NMR）分析表明，产物中的氘位于亲r位置，表明水的消除发生在C3位构型反转，这对于依赖plp的脱水酶/脱氨酶来说是前所未有的。根据晶体结构和核磁共振数据，提出了DGL的反应机理。
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.
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DOI：
10.1107/s0907444905036693
发表时间：
2006-01-01
影响因子：
2.2
作者：
Evans, P
通讯作者：
Evans, P
DOI：
10.1073/pnas.71.10.3888
发表时间：
1974-01-01
影响因子：
11.1
作者：
DUNATHAN, HC;VOET, JG
通讯作者：
VOET, JG
影响因子：
3.5
作者：
IWAMOTO, R;IMANAGA, Y;SODA, K
通讯作者：
SODA, K
DOI：
10.1107/s0907444903018043
发表时间：
2003-11-01
期刊：
ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY
影响因子：
--
作者：
Grosse-Kunstleve, RW;Adams, PD
通讯作者：
Adams, PD
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K