Molecular Mechanism Of Biosynthesis Biotin In Bacterial

The biosynthesis of biotin by microorganisms mainly goes through two stages:first stage is synthesis the pimeloyl-Co A/ACP,second stage is synthesis the bicyclic ring.Compared with the first stage,the second stage is relatively conservative among different microorganisms,which mainly catalyzed by BioF,biob,bio A and BioD in turn.There are three main pathways to synthesize pimeloyl-Co A/ACP: BioC-BioH pathway,BioI-BioW pathway,and BioZ pathway.Up to now,the molecular mechanisms of the BioC-BioH pathway and BioI-BioW pathway are relatively clear,but the molecular mechanisms of BioZ pathway are still unclear.Firstly,the crystal structure of the BioJ protein of Francisella and the BioZ protein of Agrobacterium tumefaciens with the resolution of 1.58 （?） and 1.99 （?） respectively were detected by the method of protein crystal structure biology.Compared with the structure of BioH,the catalytic centers of BioJ are S151,d248 and H278.Bioj can catalyze glutaryl-ACP monoacyl ester,pimeloyl-ACP methyl ester,and pimeloyl-ACP monoacyl esterThrough the establishment of rootless evolutionary tree,we found that BioZ belongs to β-Keto-ACP polymerase family.Comparing the structure of BioZ with that of Fab H,it was found that the catalytic centers of BioZ were C115,H255 and N285.Through in vitro fatty acid synthesis system,the substrates of BioZ were identified as glutaryl-Co A/ACP and malonyl-ACP.The affinity of BioZ with glutaryl-Co A/ACP and malonyl-ACP was measured by ITC.It was found that the affinity of BioZ with malonyl-ACP was higher.Interestingly,S84,R147 and,S287 with positive charge at the bottom of BioZ reaction pocket can effectively neutralize the negative charge of carboxyl base at the end of acyl group of glutaryl-Co A/ACP,and these three amino acids play a key role in the recognition of glutaryl-Co A/ACP by BioZ.In conclusion,the crystal structures of the BioJ protein of Francisella and the BioZ protein of Agrobacterium tumefaciens were analyzed,and the reaction mechanism between the BioJ protein and the BioZ protein was identified.A molecular model for the recognition of substrate by BioZ was proposed.