折叠的II型PLP依赖酶从<i>核杆菌中起</i>丝氨酸合酶和半胱氨酸合酶的作用,Biochemistry

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折叠的II型PLP依赖酶从核杆菌中起丝氨酸合酶和半胱氨酸合酶的作用
Biochemistry ( IF 2.9 ) Pub Date : 2021-02-04 , DOI: 10.1021/acs.biochem.0c00902
Amanda L. Darbyshire ₁ , Robert G. Mothersole ₁ , Kirsten R. Wolthers ₁

Affiliation

从丝氨酸合酶（SS）具核梭杆菌为折叠类型II的吡哆醛-5'-磷酸（PLP） -依赖性酶，其催化β-更换升用水-半胱氨酸以形成升丝氨酸和H ₂ S.此，我们表明该SS也可以用作半胱氨酸合酶，催化β-更换升丝氨酸与二硫化碳以产生升-半胱氨酸和H ₂ O的前方（丝氨酸合成酶）和反向与可比的转换数和发生（半胱氨酸合酶）的反应氨基酸底物的催化效率。SS与l的反应-半胱氨酸导致瞬态形成醌型物种，这表明Cα的去质子和β-消除从硫醇盐基团的升-半胱氨酸通过逐步机制发生。相反，在SS与L反应后，在α-氨基丙烯酸酯中间体的形成中未检测到醌类物质。-丝氨酸。已显示出关键的活性位点残基D232可稳定更具化学反应性的酮烯胺PLP互变异构体，并且在正向和反向反应中还充当酸/碱催化剂。PLP和W99（酶的唯一色氨酸残基）之间的荧光共振能量转移，支持配体诱导的活性位点封闭，从而将PLP辅因子与溶剂隔离，并提高了D232的碱性。这些结果提供了新的洞察力，在核糖核酸中的氨基酸代谢，并突出了D232在PLP依赖性酶的II型折叠家族的新成员中的多重催化作用。

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A Fold Type II PLP-Dependent Enzyme from Fusobacterium nucleatum Functions as a Serine Synthase and Cysteine Synthase

Serine synthase (SS) from Fusobacterium nucleatum is a fold type II pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyzes the β-replacement of l-cysteine with water to form l-serine and H₂S. Herein, we show that SS can also function as a cysteine synthase, catalyzing the β-replacement of l-serine with bisulfide to produce l-cysteine and H₂O. The forward (serine synthase) and reverse (cysteine synthase) reactions occur with comparable turnover numbers and catalytic efficiencies for the amino acid substrate. Reaction of SS with l-cysteine leads to transient formation of a quinonoid species, suggesting that deprotonation of the Cα and β-elimination of the thiolate group from l-cysteine occur via a stepwise mechanism. In contrast, the quinonoid species was not detected in the formation of the α-aminoacrylate intermediate following reaction of SS with l-serine. A key active site residue, D232, was shown to stabilize the more chemically reactive ketoenamine PLP tautomer and also function as an acid/base catalyst in the forward and reverse reactions. Fluorescence resonance energy transfer between PLP and W99, the enzyme’s only tryptophan residue, supports ligand-induced closure of the active site, which shields the PLP cofactor from the solvent and increases the basicity of D232. These results provide new insight into amino acid metabolism in F. nucleatum and highlight the multiple catalytic roles of D232 in a new member of the fold type II family of PLP-dependent enzymes.

更新日期：2021-02-23

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