Novel poly-γ-glutamate-processing enzyme catalyzing γ-glutamyl DD-amidohydrolysis
Novel poly-γ-glutamate-processing enzyme catalyzing γ-glutamyl DD-amidohydrolysis
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DOI：
10.1263/jbb.102.60
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发表时间：
2006-07-01
影响因子：
2.8
通讯作者：
Misono, Haruo
中科院分区：
文献类型：
--
作者：
Ashiuchi, Makoto;Nakamura, Hisaaki;Misono, Haruo
枯草芽孢杆菌的 pgdS 基因产物 PgdS 以类似内肽酶的方式裂解聚-γ-谷氨酸 (PGA)。然而，其催化性能仍然不清楚。在本研究中，开发了一种使用 1-氟-2,4-二硝基苯对 PgdS 酶进行简单测定的方法，并检查了 PgdS 的一些特性，例如最佳 pH 值。该酶被硫醇修饰试剂强烈抑制，表明它在催化作用中具有必需的半胱氨酸残基。 PgdS对主要由D-谷氨酸残基组成的PGA表现出高亲和力，但对仅由L-谷氨酸残基组成的PGA(L-PGA)没有亲和力。该酶将平均分子量为1000 kDa的DL-共聚物型PGA（DL-PGA）加工成富含L-谷氨酸的高分子量片段（平均200 kDa）、富含L的PGA片段和主要由D-谷氨酸残基组成的低分子量片段（平均5 kDa）、D-片段。为了加深对PgdS酶催化特性的理解，我们分析了N端和C端区域的结构，发现D-谷氨酰残基甚至连续位于富含L的PGA片段的两端。我们的观察表明，PgdS 是一种新型内肽酶，可特异性裂解 PGA 中两个 D-谷氨酸残基之间的 γ-酰胺键，即 γ-谷氨酰 DD-酰胺水解酶。该酶可能可用于枯草芽孢杆菌 DL-PGA 的生化处理。
The pgdS gene product of Bacillus subtilis, PgdS, cleaves poly-gamma-glutamate (PGA) in an endopeptidase-like fashion. However, its catalytic property remains obscure. In this study, a simple assay for the PgdS enzyme using 1-fluoro-2,4-dinitrobenzene was developed, and some characteristics of PgdS, such as optimal pH, were examined. The enzyme was strongly inhibited by a thiol-modifying reagent, suggesting that it possesses essential cysteine residue(s) in catalysis. PgdS exhibited a high affinity to PGA that consisted mainly of D-glutamate residues, but no affinity to PGA composed only of L-glutamate residues (L-PGA). The enzyme processed DL-copolymer-type PGA (DL-PGA) with an average molecular mass of 1000 kDa to a high-molecular-mass L-glutamate-rich fragment (average 200 kDa), the L-rich PGA fragment, and low-molecular-mass fragment composed mostly of D-glutamate residues (average 5 kDa), D-fragment. To deepen our understanding of the catalytic property of the PgdS enzyme, we analyzed the structures of the N- and C-terminal regions and found that D-glutamyl residues successively lie even at both ends of the L-rich PGA fragment. Our observations indicate that PgdS is a novel endo-peptidase that specifically cleaves the gamma-amide linkage between two D-glutamate residues in PGA, i.e., gamma-glutamyl DD-amidohydrolase. The enzyme is possibly useful in the biochemical processing of B. subtilis DL-PGA.