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首页> 外文期刊>Applied Microbiology >Identification of a Specific Maleate Hydratase in the Direct Hydrolysis Route of the Gentisate Pathway
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Identification of a Specific Maleate Hydratase in the Direct Hydrolysis Route of the Gentisate Pathway

机译：在Gentisate途径的直接水解途径中特定马来酸盐水合酶的鉴定

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In contrast to the well-characterized and more common maleylpyruvate isomerization route of the gentisate pathway, the direct hydrolysis route occurs rarely and remains unsolved. In Pseudomonas alcaligenes NCIMB 9867, two gene clusters, xln and hbz , were previously proposed to be involved in gentisate catabolism, and HbzF was characterized as a maleylpyruvate hydrolase converting maleylpyruvate to maleate and pyruvate. However, the complete degradation pathway of gentisate through direct hydrolysis has not been characterized. In this study, we obtained from the NCIMB culture collection a Pseudomonas alcaligenes spontaneous mutant strain that lacked the xln cluster and designated the mutant strain SponMu. The hbz cluster in strain SponMu was resequenced, revealing the correct location of the stop codon for hbzI and identifying a new gene, hbzG . HbzIJ was demonstrated to be a maleate hydratase consisting of large and small subunits, stoichiometrically converting maleate to enantiomerically pure d-malate. HbzG is a glutathione-dependent maleylpyruvate isomerase, indicating the possible presence of two alternative pathways of maleylpyruvate catabolism. However, the hbzF -disrupted mutant could still grow on gentisate, while disruption of hbzG prevented this ability, indicating that the direct hydrolysis route was not a complete pathway in strain SponMu. Subsequently, a d-malate dehydrogenase gene was introduced into the hbzG -disrupted mutant, and the engineered strain was able to grow on gentisate via the direct hydrolysis route. This fills a gap in our understanding of the direct hydrolysis route of the gentisate pathway and provides an explanation for the high yield of d-malate from maleate by this d-malate dehydrogenase-deficient natural mutant.
机译：与龙胆酸盐途径的特征明确且更常见的马来酸丙酮酸异构化途径相反，直接水解途径很少发生且仍未溶解。在产碱假单胞菌NCIMB 9867中，先前提出了两个基因簇xln和hbz参与龙胆酸盐的分解代谢，HbzF被表征为马来酸丙酮酸水解酶，将马来酸丙酮酸转化为马来酸和丙酮酸。但是，尚未描述龙胆酸盐通过直接水解的完整降解途径。在这项研究中，我们从NCIMB培养物收集物中获得了一种缺乏xln簇的拟青霉假单胞菌自发突变株，并将其命名为SponMu突变株。对菌株SponMu中的hbz簇进行了重新测序，揭示了hbzI终止密码子的正确位置并鉴定了一个新基因hbzG。事实证明，HbzIJ是由大亚基和小亚基组成的马来酸水合酶，化学计量地将马来酸转化为对映体纯的d-苹果酸。 HbzG是谷胱甘肽依赖性的马来酸丙酮酸异构酶，表明可能存在两种马来酸丙酮酸分解代谢途径。但是，受hbzF干扰的突变体仍可在龙胆酸盐上生长，而对hbzG的破坏阻止了这种能力，表明直接水解途径不是菌株SponMu中的完整途径。随后，将d-苹果酸脱氢酶基因引入到破坏hbzG的突变体中，该工程菌株能够通过直接水解途径在龙胆酸酯上生长。这填补了我们对龙胆酸酯途径的直接水解途径的理解的空白，并为该d-苹果酸脱氢酶缺陷的天然突变体从马来酸酯中高产率的d-苹果酸提供了解释。

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