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

Abstract

Nocardioides sp. PD653 genes hcbA1, hcbA2, and hcbA3 encode enzymes that catalyze the oxidative dehalogenation of hexachlorobenzene (HCB), which is one of the most recalcitrant persistent organic pollutants (POPs). In this study, HcbA1, HcbA2, and HcbA3 were heterologously expressed and characterized. Among the flavin species tested, HcbA3 showed the highest affinity for FMN with a K d value of 0.75±0.17 µM. Kinetic assays revealed that HcbA3 followed a ping-pong bi-bi mechanism for the reduction of flavins. The K m for NADH and FMN was 51.66±11.58 µM and 4.43±0.69 µM, respectively. For both NADH and FMN, the V max and k cat were 2.21±0.86 µM and 66.74±5.91 sec −1 , respectively. We also successfully reconstituted the oxidative dehalogenase reaction in vitro, which consisted of HcbA1, HcbA3, FMN, and NADH, suggesting that HcbA3 may be the partner reductase component for HcbA1 in Nocardioides sp. PD653.

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.…”

Potential Changes in Soil Microbial Composition under 1,2-Dichlorobenzene Contamination

“…Furthermore, greater numbers of Pseudomonas, Sphingomonas, Nocardioides, Anaeromyxobacter, and unclassified genera of the Xanthomonadaceae family were found in the treatments amended with N and P concentrations. Studies indicate that many species in these genera can degrade chlorine-containing organic substances [69][70][71], and other studies show that these genera are important in nitrate reduction [72][73][74]. Thus, we suspect that the high relative abundance of these three OTUs may correlate with the presence of 1,2-DCB in the microcosms amended with nitrate.…”

Synthetic studies of biologically active natural products contributing to pesticide development

“…54) Degradation pathways of other POPs such as endosulfan and hexachlorobenzene (HCB) were also studied. [55][56][57] Fig. 8.…”