Hexachlorobenzene Catabolism Involves a Nucleophilic Aromatic Substitution and Flavin-N5-Oxide Formation

Abstract

HcbA1 is a unique flavoenzyme that catalyzes the first step in the bacterial hexachlorobenzene catabolic pathway. Here we report in vitro reconstitution of the HcbA1-catalyzed reaction. Detailed mechanistic studies provide evidence for nucleophilic aromatic substitution and flavin-N5-oxide formation.

Biochemical characterization of NADH:FMN oxidoreductase HcbA3 from <i>Nocardioides</i> sp. PD653 in catalyzing aerobic HCB dechlorination

“…Purified HcbA1 dehalogenates HCB in the presence of an Escherichia coli flavin reductase (Fre), 6) FMN, and NADH, resulting in the formation of flavin-N5-oxide. 7) These results are consistent with our hypothesis and imply the presence of a partner reductase component of HcbA1. Notably, the co-expression of hcbA1 and hcbA3 leads to HCB dehalogenase activity in E. coli cells; hence, HcbA3 is believed to supply reduced flavin in vivo.…”

“…Purified HcbA1 displays HCB dehalogenase activity with photoreduced FMN and molecular oxygen in vitro. 7) However, due to the lack of biochemical analysis of the flavin reductase component, it remained unclear whether the oxidative HCB dehalogenation was catalyzed by a TC-FDM system. Thus, the goal of the current study was to first characterize the flavin reductase component in a single-enzyme assay and then clarify whether HCB dehalogenation was catalyzed in a coupled assay with HcbA1.…”

Mechanisms of aerobic dechlorination of hexachlorobenzene and pentachlorophenol by <i>Nocardioides</i> sp. PD653

“…This feature was also found to be common to the hcbA genes, all three genes of which were transcribed polycistronically. 35) We postulated that the mechanism underlying the initial HCBdechlorination step in PD653 is closely associated with the TC-FDM system; purified HcbA1 was indeed found to dechlorinate HCB in the presence of an E. coli flavin reductase (Fre), 36) FMN, and NADH, resulting in the formation of flavin-N5-oxide, 37) thereby supporting our hypothesis and implying the presence of the partner reductase component of HcbA1. Given our observation that co-expression of hcbA1 and hcbA3 led to HCB dehalogenase activity in E. coli cells, we believe that HcbA3 plays a role in supplying reduced flavin in vivo.…”

Structures of the alkanesulfonate monooxygenase MsuD provide insight into C–S bond cleavage, substrate scope, and an unexpected role for the tetramer

“…7 ). At this point the MsuD mechanism has diverged from the general proposed mechanisms for RutA, DszA, and HcbA1 ( 35 ), in which the organic substrates and products are in the same oxidation state and the cosubstrate FMNH − forms flavin-N5-oxide; a reducing equivalent of NADH is needed to reform FMN ( 52 , 53 , 54 ). However, in MsuD the starting alkanesulfonate is more reduced than the product, formaldehyde, and the cosubstrate FMNH − stays in its reduced form after hydroxylation.…”