Active Site Structure and Mechanism of a Molybdenum Catechol Dehydroxylase

The dehydroxylation of catechols represents an important chemical transformation facilitated by gut bacteria in mammals. This reaction is catalyzed by pyranopterin molybdenum enzymes that belong to the DMSO reductase family. Despite their chemical and biological significance, the structure and mechanisms of catechol dehydroxylases remain uncharacterized. In this manuscript, we interrogated the active site structure of hydrocaffeic acid dehydroxylase from the gut bacterium Gordonibacter urolithinfaciens (Gu Hcdh) using Mo K-edge X-ray absorption near-edge structure (XANES) spectroscopy and extended X-ray absorption fine structure (EXAFS) analyses. In the oxidized state, the Mo(VI) ion is coordinated by a terminal oxo atom, a cysteine thiolate, and four sulfur atoms from the two bidentate pyranopterin dithiolene (PDT) ligands. Upon reduction to the Mo(IV) state, the active site remains hexacoordinate with a similar first coordination sphere; however, the terminal oxo ligand present in the Mo(VI) state has been protonated to yield a coordinated hydroxyl ligand. The EXAFS-derived coordination geometries for the Mo(VI) and Mo(IV) sites are consistent with the results of bond valence sum (BVS) analyses. Reaction coordinate computations suggest the likely role of an active site carboxylate in facilitating substrate dearomatization and product formation. Protein sequence analysis and site-directed mutagenesis experiments reveal that Cys157 is ligated to the Mo ion, and Asp210 serves as a catalytically essential active site acid-base. Together, these analyses enrich our understanding of an emerging pyranopterin molybdenum enzyme family from the human gut microbiota.