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Biochemical characterization of NADH:FMN oxidoreductase HcbA3 from Nocardioides 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.

SUBMITTER: Ito K

PROVIDER: S-EPMC7453296 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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

Ito Koji K Takagi Kazuhiro K Kataoka Ryota R Kiyota Hiromasa H

Journal of pesticide science 20200801 3

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 followe ...[more]

PMID: 32913414

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Project description:BackgroundTuberculosis (TB) remains an important public health problem since it is the major cause of elevated morbidity and mortality globally. Previous works have shown that Mycobacterium tuberculosis (Mtb); the prime causative agent of the deadly disease has dormancy survival regulator (DosR) regulon, a two-component regulatory system which controls the transcription of more than 50 genes. However, the structure and detailed functions of these DosR regulated genes are largely undetermined. Out of many DosR regulon genes, Rv3131 gets up regulated in hypoxic conditions and was believed to encode for a nitroreductase flavoprotein. The utilization of mycobacteria-specific model systems has greatly added to our understanding of the molecular mechanisms involved in the life cycle and pathogenesis of Mtb.ResultsIn this study the non-pathogenic mycobacterial model organism Mycobacterium smegmatis (Msmeg) was used to reveal the structure and function of MSMEG_3955; which is a homologue of Rv3131 from Mtb. Using chromatography and spectroscopy techniques it was revealed that cofactor flavin mononucleotide (FMN) was bound to flavoprotein MSMEG_3955. Consistent with the homology modelling predictions, Circular Dichroism (CD) analysis indicated that the MSMEG_3955 is composed of 39.3% α-helix and 24.9% β-pleated sheets. In contrast to the current notions, the enzymatic assays performed in the present study revealed that MSMEG_3955 was not capable of reducing nitro substrates but showed NADPH dependent FMN oxidoreductase activity. Also, gel permeation chromatography, dynamic light scattering and native acidic gels showed that MSMEG_3955 exists as a homotrimer. Furthermore, the presence of NADPH dependent FMN oxidoreductase and homotrimeric existence could be an alternative function of the protein to help the bacteria survive in dormant state or may be involved in other biochemical pathways.ConclusionMSMEG_3955 is a FMN bound flavoprotein, which exits as a trimer under in vitro conditions. There is no disulphide linkages in between the three protomers of the homotrimer MSMEG_3955. It has a NADPH dependent FMN oxidoreductase activity.

Dataset Information

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

Publications

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

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