Identifaition Of The Key Enzyme Odca For 3,5-dibromo-4-hydroxybenzoate Catabolism In Strain Pigmentiphaga Sp.h8 And Its Molecular Simulation For Substrate Docking

3,5-Dibromo-4-hydroxybenzoic acid is a common type of environmental pollutants belongs to brominated aromatic compound,which poses a serious threat to human and ecological health.However,few studies on the microbial degradation of DBHB has been done.There are only two types of DBHB degradation pathways（reductive debromination and decarboxylation）have been reported so far.One is based on metabolic pathways.The other one is based on molecular interactions of gene BhbA/BhbA2/BhbA3-mediated reduction bromine.It is of great scientific significance to explore new DBHB microbial degradation pathway,Clone new degradation genes and clarify its enzymatic catalytic mechanisms.Pigmentiphaga sp.H8 is a strain isolated from long-term contaminated soil with halogenated aromatic compounds.It can mineralize 0.2 mM DBHB completely within 24 hours as their sole carbon and energy source.Induced degradation experiments showed that expression of enzymes that related to the degradation of DBHB of strain H8 was induced by DBHB.So we can find genes involved in the degradation of DBHB by comparing the differences in mRNA transcription and proteomic expression between DBHB-induced and non-induced strains with transcriptomics and comparative proteomics techniques.The results show that orf420-orf426 of the strain H8 genome may involved in degradation DBHB.Compairing proteins encoded by orf420-orf426 to the protein database we found that protein OdcA encoded by orf420（gene odcA）has the similarity of 34%with HspB in strain Pseudomonas putida S16,HspB uses NADH as electron donor and H donor to oxidize tonicotine metabolic intermediate 2,5-dihydroxypyridine to 6-hydroxy-3-succinylpyridine.We certificated the function of gene odcA by gene knockout and complementary experiments and found that it was involved in the initial degradation of DBHB.So we constructed strain E.coli BL21（DE3）pET-odcA to express OdcA which carries a N-terminal Strep II tag so that we can purified it by Strep-Tactin Sepharose HP affinity chromatography.By in vitro enzymatic transformation experiments and LC-MS analysis,we identified the product of OdcA degradation of DBHB was 2,6-dibromohydroquinone.Amino acid sequence analysis showed that OdcA was a hydrophobic protein consisting of 402 amino acids with a molecular weight of 44.6 kDa and a theoretical isoelectric point of 5.25.The 4th to 348th amino acids form an FAD/NAD（P）binding domain.The degradation of DBHB catalyzed by OdcA requires the involvement of cofactor NADPH or NADH,and NADPH is about 1.5 times that of NADH catalytic activity.The best enzymatic reaction temperature of OdcA is 35℃,30-40℃ range can also show good enzyme activity;the optimum enzymatic reaction pH is 7;0.1 mM Mn2+ can strongly inhibit OdcA enzyme activity,0.1 mM Cu2+,Ca2+,Fe3+,Fe2+,Co3+ and Ni2+ had a slight inhibitory effect on OdcA,while 0.1 mM Zn2+ could slightly promote OdcA’s activity.The Km value of OdcA for DBHB was 39.3 ± 5.1 mM,the Vmax was 4.1±0.2 μM·min^-1,and the Kcat value was 35.9μM·min^-1 at 35℃,with NADPH as the electron donor.Prediction of the three-dimensional structure of OdcA and the docking simulation between OcdA and DBHB showed that No.107 GLN（Glutamine）of OdcA interacted with carbonyl（-CO-）in DBHB molecule,which indicated that O2 molecules may attack the carbonyl group,so that oxygen decarboxylation to generate CO2.These results are consistent with the enzymatic function of the OdcA-it catalyzed DBHB decarboxylation by oxidation to produce 2,6-dibromohydroquinone.In general,this study focused on a key gene ocdA of metabolic pathway of DBHB in strain H8,and elaborated a new degradation mechanism at the enzymic level.This not only enriches the theoretical basis of the biodegradation of halobenzoic acid,but also provides new genes and enzyme resources for the biodegradation and remediation of halogenated aromatics.