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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. InPseudomonas alcaligenes NCIMB 9867, two gene clusters,xln andhbz , 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 aPseudomonas alcaligenes spontaneous mutant strain that lacked thexln cluster and designated the mutant strain SponMu. Thehbz cluster in strain SponMu was resequenced, revealing the correct location of the stop codon forhbzI 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 pured -malate. HbzG is a glutathione-dependent maleylpyruvate isomerase, indicating the possible presence of two alternative pathways of maleylpyruvate catabolism. However, thehbzF -disrupted mutant could still grow on gentisate, while disruption ofhbzG prevented this ability, indicating that the direct hydrolysis route was not a complete pathway in strain SponMu. Subsequently, ad -malate dehydrogenase gene was introduced into thehbzG -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 ofd -malate from maleate by thisd -malate dehydrogenase-deficient natural mutant.

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