Identification of a Specific Maleate Hydratase in the Direct Hydrolysis Route of the Gentisate Pathway

Abstract

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. G entisate (2,5-dihydroxybenzoate) is an important ring cleavage intermediate present in the bacterial catabolic pathway of many aromatic compounds. In the gentisate pathway, gentisate 1,2-dioxygenase catalyzes the ring cleavage oxidation of gentisate to yield maleylpyruvate, which is then further degraded by either direct hydrolysis to maleate and pyruvate (1) or isomerization to fumarylpyruvate and subsequent hydrolysis to fumarate and pyruvate (2, 3). For the isomerization route, glutathione (GSH)-, mycothiol (MSH)-, or L-cysteine-dependent maleylpyruvate isomerases have been biochemically and genetically characterized in Gram-negative (4), high-GϩC-content Gram-positive (5, 6), or low-GϩC-content Gram-positive (7) bacterial strains, respectively. As an alternative to the isomerization route, maleylpyruvate can also be degraded via the direct hydrolysis route, which is catalyzed by maleylpyruvate hydrolase to yield maleate and pyruvate (Fig. 1). In contrast to the well-studied isomerization route, the direct hydrolysis route has been studied only in the 2,5-and 3,5-xylenol utilizer Pseudomonas alcaligenes NCIMB 9867 (8, 9), in which m-cresol, xylenols, and 3-hydroxybenzoate are degraded via gentisate. In this strain, a maleylpyruvate hydrolaseencoding gene, hbzF, was ch...

A Novel Degradation Mechanism for Pyridine Derivatives in Alcaligenes faecalis JQ135

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Combining Electro-, Photo-, and Biocatalysis for One-Pot Selective Conversion of Furfural into Value-Added C4 Chemicals

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