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 make up the largest group of characterized extradiol dioxygenases, and while most are found as part of catabolic pathways degrading a variety of natural and human-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, yet despite decades of investigation of 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 its evolutionary similarity to glyoxylase enzymes, we performed a careful investigation of the Streptomyces 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 the existing knowledge of VOC extradiol dioxygenase catalysis.

Dioxygenase enzymes are essential protein catalysts for the breakdown of catecholic rings, structural components of plant woody tissue. This powerful chemistry is used in nature to make antibiotics and other bioactive materials or degrade plant material, but we have a limited understanding of the breadth and depth of substrate space for these potent catalysts. Here we report steady-state and pre-steady-state kinetic analysis of dopamine derivatives substituted at the 6-position as substrates of L-DOPA dioxygenase, and an analysis of that activity as a function of the electron-withdrawing nature of the substituent. Steady-state and pre-steady-state kinetic data demonstrate the dopamines are impaired in binding and catalysis with respect to the cosubstrate molecular oxygen, which likely afforded spectroscopic observation of an early reaction intermediate, the semiquinone of dopamine. The reaction pathway of dopamine in the pre-steady state is consistent with a nonproductive mode of binding of oxygen at the active site. Despite these limitations, L-DOPA dioxygenase is capable of binding all of the dopamine derivatives and catalyzing multiple turnovers of ring cleavage for dopamine, 6-bromodopamine, 6-carboxydopamine, and 6-cyanodopamine. 6-Nitrodopamine was a single-turnover substrate. The variety of substrates accepted by the enzyme is consistent with an interplay of factors, including the capacity of the active site to bind large, negatively charged groups at the 6-position and the overall oxidizability of each catecholamine, and is indicative of the utility of extradiol cleavage in semisynthetic and bioremediation applications.