A Novel Bifunctional Amino Acid Racemase With Multiple Substrate Specificity, MalY From Lactobacillus sakei LT-13: Genome-Based Identification and Enzymological Characterization.
A Novel Bifunctional Amino Acid Racemase With Multiple Substrate Specificity, MalY From Lactobacillus sakei LT-13: Genome-Based Identification and Enzymological Characterization.
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DOI：
10.3389/fmicb.2018.00403
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发表时间：
2018
影响因子：
5.2
通讯作者：
Oikawa T
中科院分区：
文献类型：
--
作者：
Kato S;Oikawa T
从日本清酒的发酵剂Moto分离的清酒乳杆菌菌株LK-145在含有淀粉酶消化的大米作为碳源的培养基中潜在地产生大量的三种D-氨基酸，D-Ala、D-Glu和D-Asp。从菌株LK-145的全基因组序列推导的代谢途径的比较，清酒乳杆菌菌株LT-13的典型培养物菌株表明，L-和D-氨基酸代谢途径是相似的两个菌株之间。然而，在菌株LK-145和LT-13的推定半胱氨酸/甲硫氨酸代谢途径中观察到显著差异。胱硫醚β-裂解酶同源基因malY仅在菌株LT-13的基因组中被注释。胱硫醚β-裂解酶是半胱氨酸/甲硫氨酸代谢途径中催化L-胱硫醚转化为L-同型半胱氨酸的重要酶。除malY外，大多数基因组测序的L.包括LT-13在内的清酒缺乏编码该途径中其他推定酶的同源基因。因此，半胱氨酸/甲硫氨酸代谢途径可能在几乎所有的L.清酒。本研究成功地克隆了LT-13菌株的malY基因（Ls-malY），并在大肠杆菌BL 21（DE 3）中表达，同时对Ls-MalY的酶学性质进行了研究。对纯化的Ls-MalY进行光谱分析，结果表明Ls-MalY中含有一个5′-磷酸吡哆醛（PLP）作为辅因子，这一发现与基于其一级结构的预测相一致。Ls-MalY具有氨基酸消旋酶活性和胱硫醚β-裂解酶活性。Ls-MalY对Ala、Arg、Asn、Glu、Gln、His、Leu、Lys、Met、Ser、Thr、Trp和瓦尔等多种氨基酸均具有消旋酶活性。突变分析表明Ls-MalY一级结构中Lys 233的N-氨基可能与PLP结合，Lys 233是Ls-MalY催化氨基酸消旋酶和β-裂解酶反应的必需残基。此外，Tyr 123在氨基酸消旋酶反应中是一个催化残基，但对β-裂解酶活性有很大影响。这些结果表明，Ls-MalY是一种新型的双功能氨基酸消旋酶，具有多底物特异性，其氨基酸消旋酶和β-裂解酶反应均在同一活性位点催化。
The Lactobacillus sakei strain LK-145 isolated from Moto, a starter of sake, produces potentially large amounts of three D-amino acids, D-Ala, D-Glu, and D-Asp, in a medium containing amylase-digested rice as a carbon source. The comparison of metabolic pathways deduced from the complete genome sequence of strain LK-145 to the type culture strain of Lactobacillus sakei strain LT-13 showed that the L- and D-amino acid metabolic pathways are similar between the two strains. However, a marked difference was observed in the putative cysteine/methionine metabolic pathways of strain LK-145 and LT-13. The cystathionine β-lyase homolog gene malY was annotated only in the genome of strain LT-13. Cystathionine β-lyase is an important enzyme in the cysteine/methionine metabolic pathway that catalyzes the conversion of L-cystathionine into L-homocysteine. In addition to malY, most genome-sequenced strains of L. sakei including LT-13 lacked the homologous genes encoding other putative enzymes in this pathway. Accordingly, the cysteine/methionine metabolic pathway likely does not function well in almost all strains of L. sakei. We succeeded in cloning and expressing the malY gene from strain LT-13 (Ls-malY) in the cells of Escherichia coli BL21 (DE3) and characterized the enzymological properties of Ls-MalY. Spectral analysis of purified Ls-MalY showed that Ls-MalY contained a pyridoxal 5′-phosphate (PLP) as a cofactor, and this observation agreed well with the prediction based on its primary structure. Ls-MalY showed amino acid racemase activity and cystathionine β-lyase activity. Ls-MalY showed amino acid racemase activities in various amino acids, such as Ala, Arg, Asn, Glu, Gln, His, Leu, Lys, Met, Ser, Thr, Trp, and Val. Mutational analysis revealed that the 𝜀-amino group of Lys233 in the primary structure of Ls-MalY likely bound to PLP, and Lys233 was an essential residue for Ls-MalY to catalyze both the amino acid racemase and β-lyase reactions. In addition, Tyr123 was a catalytic residue in the amino acid racemase reaction but strongly affected β-lyase activity. These results showed that Ls-MalY is a novel bifunctional amino acid racemase with multiple substrate specificity; both the amino acid racemase and β-lyase reactions of Ls-MalY were catalyzed at the same active site.
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影响因子：
--
作者：
Lowther, Jonathan;Beattie, Ashley E.;Campopiano, Dominic J.
通讯作者：
Campopiano, Dominic J.
影响因子：
3.2
作者：
Mutaguchi, Yuta;Ohmori, Taketo;Ohshima, Toshihisa
通讯作者：
Ohshima, Toshihisa
DOI：
10.1073/pnas.0808269106
发表时间：
2009-01-20
影响因子：
11.1
作者：
Li, Congran;Lu, Chung-Dar
通讯作者：
Lu, Chung-Dar
影响因子：
--
作者：
Mutaguchi Y;Ohmori T;Akano H;Doi K;Ohshima T
通讯作者：
Ohshima T
DOI：
10.1128/genomea.00656-17
发表时间：
2017-08-01
期刊：
GENOME ANNOUNCEMENTS
影响因子：
--
作者：
Kato, Shiro;Oikawa, Tadao
通讯作者：
Oikawa, Tadao