Molecular Basis of BioJ, a Unique Gatekeeper in Bacterial Biotin Synthesis

Abstract

SummaryBiotin is an indispensable cofactor in the three domains of life. The unusual virulence factor BioJ of Francisella catalyzes the formation of pimeloyl-ACP, an intermediate in biotin synthesis. Here, we report the 1.58 Å crystal structure of BioJ, the enzymatic activity of which is determined with the in vitro reconstituted reaction and biotin bioassay in vivo. Unlike the paradigm BioH, BioJ displays an atypical α/β-hydrolase fold. A structurally conserved catalytic triad (S151, D248, and H278) of BioJ is functionally defined. A proposed model for BioJ catalysis involves two basic residues-rich cavities, of which cavity-1, rather than cavity-2, binds to the ACP moiety of its physiological substrate, pimeloyl-ACP methyl ester. In summary, this finding provides molecular insights into the BioJ gatekeeper of biotin synthesis.

Characterization of the Bifunctional Enzyme BioDA Involved in Biotin Synthesis and Pathogenicity in Aspergillus flavus

“…Growth curves were performed as described before. 68 First, the strains were grown in a liquid M9 minimal medium containing 1 nM biotin at 30 °C overnight. The strains were harvested by centrifugation at 4000 r/min, and then the pellet was washed with M9 buffer lacking biotin three times.…”

“…Expression, purification, and size exclusion chromatography were carried out as described before with minor changes. 68 The PCR product of bioDA amplified from the cDNA of A. flavus was cloned into pET28a and used for protein overexpression. Bacterial culture induced with 0.5 mM IPTG was harvested by centrifugation at 5000 r/min for 15 min.…”

“…Growth curves were performed as described before . First, the strains were grown in a liquid M9 minimal medium containing 1 nM biotin at 30 °C overnight.…”

“…Expression, purification, and size exclusion chromatography were carried out as described before with minor changes . The PCR product of bioDA amplified from the cDNA of A.…”

Three enigmatic BioH isoenzymes are programmed in the early stage of mycobacterial biotin synthesis, an attractive anti-TB drug target

“…The BioC-aided methylation enables the resultant malonyl-ACP (CoA) methyl ester to undergo two reiterations of FAS II cycles [ 32 ], generating methyl pimeloyl-ACP thioester that is subsequently hydrolyzed by the promiscuous esterase BioH to give pimeloyl-ACP (pim-ACP or C7-ACP), a precursor for biotin synthesis [ 23 , 31 ]. To the best of our knowledge, six isoenzymes of BioH have been detected in various bacterial species, namely BioG [ 38 , 39 ], BioK [ 38 ], BioJ [ 40 , 41 ], BioV [ 42 ], BtsA [ 43 ], and BioUh [ 22 , 44 ]. As for ‘BioI-BioW’ model of Bacillus subtilis , the cytochrome P450 protein BioI presumably cleaves long-chain fatty acids (and/or acyl-ACP) to give pimeloyl-ACP [ 35 , 45 ], whereas the bona fide acyl-CoA synthetase BioW scavenges exogenous pimelate to yield pimeloyl-CoA thioester [ 33 , 34 , 46 ], a substrate specifically recognized by the Bacillus BioF [ 25 , 45 ].…”