Identification And Functional Analysis Of O-methyltransferases In Streptomyces Virginiae IBL14

Streptomyces virginiae IBL14is an effective degradative strain for various flavonoid compounds and can methoxylate diosgenin to6-methoxy-products, the first-ever bio-methoxylation on the B-ring of steroidal compounds, discovered in our lab.To interpret the mechanism of6-methoxylation on the B-ring of diosgenin by S. virginiae IBL14, the whole genome has been sequenced. The analyses of Basic Local Alignment Search Tool-Protein （BLASTP） based on the data of the genome sequencing demonstrated that the strain IBL14contains9putative O-methyltransferases （OMTs, EC2.1.1.X） in its8.0Mb linear chromosome.The results of analysis by software indicated that the nine putative OMTs of S. virginiae IBL14belong to methyltransf₂superfamily （SviOMT01, SviOMT05and SviOMT08）, amdomet-MTases superfamily （SviOMT02, SviOMT03, SviOMT04, SviOMT06and SviOMT07） and leucine carboxyl methyltransferase （LCM） superfamily （SviOMT09）. From the topology of the phylogenetic tree, S. virginiae IBL14have the closest relationships to those of Streptomyces sp. Mgl and Streptomyces sp. C., it also reveal that SviOMT03and SviOMT06have a close affinity with CCOMTs from Streptomyces spp, suggesting that SviOMT03and SviOMT06might be able to biotransform caffeoyl-CoA and/or caffeic acid into feruloyl-CoA and/or ferulic acid.In this paper, all of the putative O-methyltransferases gene from the strain IBL14were cloned into E.coil. The corresponding recombinant strains were analyzed by gel electrophoresis and further confirmed by sequencing, and induced by IPTG to express the protein. To identify the functions of expressed proteins:SviOMT02, SviOMT03and SviOMT06, substrates catechol,3,4-dihydroxybenzoic acid,3,4-dihydroxyphenylacetic acid, caffeic acid and3-hydroxycinnamicacid were added into the cultures of the recombinant strains E. coli IBL16M2, IBL16M3and1BL16M6as well as E. coli JM109DE3) and E. coli JM109（DE3）/pET22b as a control. As to the analysis results of the phylogenetic tree,1BL16M3and IBL16M6, as we expected, can bio transform caffeic acid into ferulic acid, Furthermore, IBL16M6can biotransform3,4-dihydroxybenzoic acid into4-hydroxy-3-methoxybenzoic acid.To analysis the interaction function between protein and small molecular substrates via the Homology modeling and molecular docking, we found out that the reaction between the polar amino acid residue Lysl43and caffeic acid as well as the reaction between Glu38（SviOMT03）/Ser44（SviOMT06）, Ser74, Asp140, Tyr149and SAM are essential in their O-methylation. It has been reported that in the crystal structure of animal COMTs, two pockets, i.e., two active sites accommodate the adenosine and methionine side chains of SAM. The two active sites display two main conformations of COMT:one open state and one closed state. Only the open state of COMT in the presence of SAM permits SAM access the active site, then transferring the methyl group from SAM to catechol. Further analyses suggested that the3-hydroxyl proton of the substrate caffeic acid might have been transferred to the nitrogen atom of His142（SviOMT03） or His171（SviOMT06）.While the active site for SAM access is in an open state and the substrate caffeic acid resides the hydrophilic domain, the phenolate ion （strong nucleophile） directly extracts the methyl group from the positively charged Sd of SAM to produce ferulic acid, and the proton at the nitrogen atom of His142or His171transfers to SAM to from SAH, in a single SN2reaction.According to the result of the conservatism of the protein and molecular docking, selecting Ser74as a mutation in the protein targets, turning it into Ala, identify the mutation of protein function:mutation protein can not transform caffeic acid into ferulic acid...