A New Way of Belonging: Active-Site Investigation of L-DOPA Dioxygenase, a VOC Family Enzyme from Lincomycin Biosynthesis

Abstract

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.

L-DOPA Dioxygenase Activity on 6-Substituted Dopamine Analogues

“…Docking was performed using the L-DOPA dioxygenase structure from Streptomyces sclerotialus, a close homologue of the S. lincolnensis enzyme. 9,26 The Fe II charge at the active site and charges on the dopamine derivatives were computed with ANTECHAMBER 36 prior to using the structures in docking. Docks were assessed by comparison to the experimentally visualized L-DOPA substrate included in the S. sclerotialus L-DOPA dioxygenase structure and through measurement of contacts to the active site Fe II and the catalytic residues His74 and Tyr144.…”

“…The Fe II content of purified or reconstituted L-DOPA dioxygenase was measured using a calibrated Ferrozine assay as previously described. 9 All concentration measurements were taken in triplicate, and errors were calculated as standard deviations.…”

“…For example, L-DOPA dioxygenase (NCBI CAA5574.1) is a member of the VOC superfamily. It is characterized by a distinct topology and domain organization, 9 and it is present in nature as a part of a biosynthetic pathway to the antibiotic lincomycin, taking the catechols of nature and recycling them into useful materials.…”

“…In an effort to improve our understanding of substrate diversity and mechanism across the VOC superfamily, we undertook the study of L-DOPA dioxygenase, a VOC dioxygenase of type IV topology, 9 and performed a structurally informed 26 examination of the cleavage reaction across a suite of electronically differentiated substrates, while considering oxygen as the co-substrate. The oxygen solubility and diffusion rate in aqueous solutions are poor, making the reactivity with oxygen an essential component of this study.…”

“…However, the extent to which this mechanism applies across the VOC superfamily and beyond is limited by our understanding of that mechanism as it operates on a variety of substrates and across different superfamily members. For example, in addition to the limited suite of substrates examined for mechanism, the mechanistic work available in the VOC superfamily has been performed only on enzymes of form V topology, , enzymes that represent only a fraction of the EDX diversity within the VOC superfamily.…”