Functional Identification And Catalytic Mechanism Of BioF In Biotin Synthesis Pathway Of Mycobacteria

Mycobacterium tuberculosis （Mtb） is one of the most important human pathogens, which can cause the TB and is a serious threat to human health. During recent years, the emergence and spread of MDR （Multi-drug resistant） and XDR （Extensive-drug resistant） Mtb and the Mtb/HIV co-infection has made the deadly TB coming back as a worldwide epidemic and prevented step for people to treat and defeat tuberculosis finally, which makes the development of new anti-TB drugs become particularly important and urgent.Biotin （aka vitamin H or B7） is an essential cofactor for some essential biotin-dependent enzymes such as pyruvate carboxylase （PC） and acyl-CoA carboxylase （ACC） and so on and function in carboxylation, decarboxylation, and transcarboxylation reactions found in many processes such as fatty acid biosynthesis, gluconeogenesis, and amino acid metabolism. Biotin synthetic pathway can be divided into two parts, the synthesis of precursor molecule pimeloyl-CoA （or pimeloyl-ACP） and the synthesis of biotin from pimeloyl-CoA （or pimeloyl-ACP）. The first part has not been well described, but it has been revealed that the synthesis pathway is varying between different species. The process of the second part, from pimeloyl-CoA （or pimeloyl-ACP） to biotin, can be divided into four steps, catalyzed by BioF, BioA, BioD and BioB, respectively. The four step reactions are very conservative in all the organisms including the biotin biosynthetic pathway.The study has suggested that biotin is an essential cofactor required for synthesis of the fatty acid component of Mtb’s cell envelope. However, only plants and microorganisms can synthesize biotin by themsmlves, whereas mammals must obtain it from external sources. Therefore biotin biosynthesis pathway has been considered as a potential target for the development of new antibiotics. The study has revealed that biotin synthesis pathway is essential for the growth and infection of M. tuberculosis. Though people have found two inhibitors, amiclenomycin and acidomycin, aim at biotin synthesis pathway, especially against BioA, but both of them were demonstrated to be inactive in a mouse model of tuberculosis. 8-Amino-7-oxononanoate Synthase （BioF） is the enzyme involving in the first committed step of biotin synthesis pathway by catalyzing the decarboxylative condensation of L-alanine with pimeloyl-CoA to form8（S）-amino-7-oxononanoate with pyridoxal5’-phosphate （PLP） as cofactor and play an key role as the gatekeeper of the path way. The genome sequence of M. tuberculosis contains two bioF genes （bioFl, Rv1569and bioF2, Rv0032）, and the proteins encoded by them are MtBioFl and MtBioF2respectively. MtBioF2consists of not only the BioF domain but an acetyltransferase domain. The genome sequence of M. smegmatis contains only one bioF gene （MSMEG₃189）, the protein which encodes is named MsBioF, with sharing76%identity with the M. tuberculosis protein MtBioFl and35%identity with the BioF domain of MtBioF2. The importance of mycobacterial bioFl has been widely recognized and confirmed, Previous study using the method of transposon site hybridization has showed that MtbioFl was essential not only for the in vitro growth but also for the growth and establish infection in vivo. But the structural properties and catalytic reaction mechanism of MtBioF1is unclear and requires further study.As mentioned above, MtBioFl is a potential target for development of anti-tuberculosis drugs. There is high identity between MsBioF and MtBioF1. Also, there are unique advantages with M. smegmatis as a model organism of mycobacteria. Thus, our study is mainly focus on the MsBioF in M. smegmatis which is usually researched as the model of mycobateria. The main results are as follows:1、We expressed and purified the high quality MsBioF protein using expression system of E. coli. The physicochemical property of MsBioF was analysed.To identify the physical and chemical properties of MsBioF, we purified the MsBioF protein. Then we firstly identified the protein sequence of MsBioF with Maldi-TOF mass spectrometry. When MsBioF is characterized by gel filtration analysis and analytical ultracentrifugation, the result shown that the oligomeric state of the protein was monomer （44.5kDa） which is different from the homologue protein AONS in E. coli. What’s more, as a PLP-dependent enzyme, MsBioF should bind PLP covalently, but Q-TOF mass spectrometry analysis showed that MsBioF could not form the adduct with PLP in vitro which might be explained by that MsBioF in monomeric condition did not have the ability to bind PLP. The study revealed that there was distinct difference between MsBioF and E. coli AONS. Meanwhile, we cut the His tag of purified MsBioF with the TEV protease and used it to immunized mouse. Fianally, we identified the expression of MsBioF in vivo with Western blot.2、The crystal of MsBioF was screened and optimized until its diffraction data be good enough to collect. We then resolved the crystal structure of MsBioF and analyzed its structural characteristic.We optimized previous purified method of MsBioF and obtained the MsBioF protein which was good enough to screen the crystal of the protein. We screened three conditions in which the crystal was growing. Then we optimized the three conditions in PH, salinity and concentration of precipitant, respectively. The crystal structure of MsBioF was resovled in a2.3A resolution. The three-dimensional structure of MsBioF revealed that each asymmetric unit of the cell contained four monomers, with differing from in solution. The structrue indicated that the enzyme forms a symmetric homodimer within the crystal. Superimposition of the MsBioF and E. coli AONS structures reveals several significant differences in overall structure. First, the whole structure of MsBioF is constrictive comparing to E.coli AONS except a6, α7and α13（corresponding to a6, a7and α12）. Second, helix α5and α13are obviously longer, but α1and α11is relatively shorter than the corresponding helices α1and α10of AONS. Third, the difference between the helices length of MsBioF and AONS results in the different orientation of loops between them.3、We deleted the MsbioF gene in M. smegmatis by the method of two-step exchange with unmarked gene. The result showed that MsbioF is essential for the biotin synthesis pathway of M. smegmatis and revealed that the function of MsbioF was come ture through infecteing the biotined of biotin-dependent enzyme. In order to study the function and enzymatic mechanism of MsBioF, the unmarked M. smegmatis bioF deletion strain was constructed using the plasmids p1NIL and pGOAL. We determined the growth curve of M. smegmatis AbioF in the presence or absence of biotin. These results all showed that the bioF played an essential role in the growth and survival of M. smegmatis without exogenous biotin. We also determined the minimal concentration of biotin for the normal growth ofâ–³bioF. We also determined the minimal concentration of biotin for the normal growth ofâ–³bioF. When the concertration of biotin was lower than5nM,â–³bioF was with little to no growth, and only when it was higher than50nM which was ten times higher than biotin concentration in humanbody,â–³bioF could be growth normally. Meanwhile, to confirm that the phenotype of M. smegmatis AbioF was caused by the decrease of biotined level of some essential biotin-dependent enzyme, we identified the expression of biotin-dependent enzyme with Western blot and found that the expression of biotin-dependent enzyme AccA3reduces obviously comparing to RpoA. The result revealed that the MsbioF play the role through infecteing the biotined of biotin-dependent enzyme.4、Through sequence alignment and structure superposition, we revealed the putative catalytic site of MsBioF. We also studied the function in vivo of these active site and truncated body of MsBioF.To elucidate the catalytic reaction mechanism of MsBioF, we identified the putative catalytic site through sequence alignment and structure superposition. We constructed the complementary plasmids of MsBioF containing site mutant and substituted these residues with alanine respectively. We then identified the dependence of the growth of these meroploid strains containing site mutant on exogenous biotin. The result showed that His129, Asp200and Lys235are essential for biotin synthesis. Glu171has a significant influence on but is not essential for activity of MsBioF. And the mutations of His203have no inflence on MsBioF activity. Meanwhile, to confirm the active state of MsBioF involved in biotion synthesis in vivo, we determined the activity of truncated MsBioF cutting off1-37residues. The result shown that truncated MsBioF expression could not complement the MsbioF deletion mutant and this revealed that the N-terminal small domain was essential for activity of MsBioF and then confirmed the active state of MsBioF is dimer. What’more, base on the structural and functional analysis of MsBioF’s catalytic site, we proposed that residue Glu171and Cys136may play a regulatory rule in MsBioF’s bioactivity.5、The phenotype of M. smegmatis bioF deletion was complemented by the expression of MtbioFl, but not MtbioF2, suggesting bioF plays a conserved role in mycobacterial biotin synthesis. The result also revealed that MsBioF and MtBioFl were conserved not only in function in vivo but the structure.To determine which bioF of Mtb play a role in biotin synthesis pathway, we assessed whether the MtbioFl gene and MtbioF2gene could complement M. smegmatisâ–³bioF. MtbioFl restored the independence of exogenous biotin for M. smegmatis bioFâ–³bioF, but MtbioF2gene could not. The result showed that MtBioF1played a role in in biotin synthesis pathway. The exact function of BioF2is still to reveal. Then we arduously purified the MtBioF1and the analysis of gel filtration analysis and analytical ultracentrifugation both showed that MtBioF was in a state of monomer as the same as MsBioF. And the sequence analysis of MtBioF1and MsBioF showed that the catalytic sites （His134, Asp205, Lys239in MtBioFl） and regulation sites （Cys140and Asp176） were conserved. The experiment in vivo also suggested that MtBioFl and MsBioF were conserved in the structure and function.