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A new way of belonging: active-site investigation of L-DOPA dioxygenase, a VOC family enzyme from lincomycin biosynthesis.

Extradiol dioxygenase chemistry is essential for catechol breakdown. The largest natural reservoir of catechols, or 1,2-dihydroxybenzenes, is the plant woody-tissue polymer, lignin. Vicinal-oxygen-chelate (VOC) dioxygenases are the largest group of characterized extradiol dioxygenases, and while most are found as part of catabolic pathways degrading a variety of natural and man-made aromatic rings, L-DOPA (L-3,4-dihydroxyphenylalanine) dioxygenase is a VOC enzyme that participates in the biosynthesis of a natural product. All VOC superfamily members shared conserved elements of catalysis, and yet despite decades of investigation on VOC enzymes, the relationships between VOC domain architecture and enzymatic function remain complex and poorly understood. Herein, we present evidence that L-DOPA dioxygenase is the representative member of a new topological class of VOC extradiol dioxygenases. Guided by evolutionary similarity to glyoxylase enzymes, we performed a careful investigation of the S. lincolnensis L-DOPA dioxygenase (LmbB1) active site through mutagenesis, kinetic and pH studies. Our results demonstrate that the L-DOPA dioxygenase reaction depends upon an active site tyrosine and histidine and is remarkably resilient to mutation, even at the iron-ligating residues. Evaluation of the cleavage reaction as a function of pH supports the role of a histidine in acid-base catalysis. The active-site architecture is functionally consistent with existing knowledge of VOC extradiol dioxygenase catalysis.