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

Abstract

Tuberculosis (TB) is one of the leading infectious diseases of global concern, and one quarter of the world’s population are TB carriers. Biotin metabolism appears to be an attractive anti-TB drug target. However, the first-stage of mycobacterial biotin synthesis is fragmentarily understood. Here we report that three evolutionarily-distinct BioH isoenzymes (BioH1 to BioH3) are programmed in biotin synthesis of Mycobacterium smegmatis. Expression of an individual bioH isoform is sufficient to allow the growth of an Escherichia coli ΔbioH mutant on the non-permissive condition lacking biotin. The enzymatic activity in vitro combined with biotin bioassay in vivo reveals that BioH2 and BioH3 are capable of removing methyl moiety from pimeloyl-ACP methyl ester to give pimeloyl-ACP, a cognate precursor for biotin synthesis. In particular, we determine the crystal structure of dimeric BioH3 at 2.27Å, featuring a unique lid domain. Apart from its catalytic triad, we also dissect the substrate recognition of BioH3 by pimeloyl-ACP methyl ester. The removal of triple bioH isoforms (ΔbioH1/2/3) renders M. smegmatis biotin auxotrophic. Along with the newly-identified Tam/BioC, the discovery of three unusual BioH isoforms defines an atypical ‘BioC-BioH(3)’ paradigm for the first-stage of mycobacterial biotin synthesis. This study solves a long-standing puzzle in mycobacterial nutritional immunity, providing an alternative anti-TB drug target.

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“…This enzyme has a putative role in the biosynthesis of biotin 48 and the absence of viable mutants within the Manoil Lab transposon mutant library 45 suggests that it might be essential. Of note, in Mycobacterium tuberculosis, biotin metabolism is already considered a viable antibacterial drug target pathway 83 84 , and efforts to target its three bioH isoenzymes are underway 85 . For ABPP, enzyme targets are identified through covalent interaction with a small molecule probe, thus focusing the set of targets that are often likely to be druggable and provides direct assays for development of target-specific inhibitors.…”

“…Another potential drug target that was identified in our dataset but was not validated further, is the carboxylesterase Bio H. This enzyme has a putative role in the biosynthesis of biotin 48 and the absence of viable mutants within the Manoil Lab transposon mutant library 45 suggests that it might be essential. Of note, in Mycobacterium tuberculosis, biotin metabolism is already considered a viable antibacterial drug target pathway 83,84 , and efforts to target its three bioH isoenzymes are underway 85 .…”

The opportunistic pathogen Pseudomonas aeruginosa exploits bacterial biotin synthesis pathway to benefit its infectivity

“…The advantage of this genetic arrangement is to prevent wasteful production of two enzymes dedicated to the primary stage of biotin synthesis. Whereas a rare example of exception was recently discovered in Mycobacterium smegmatis , i.e., three BioH isoforms (BioH1 to BioH3) combined with a single BioC, are programed into an early stage of mycobacterial biotin synthesis [ 62 ]. Presumably, the redundance of BioH-like activities is originated from the domestication of certain α/β-hydrolase to gain this promiscuous role of hydrolyzing M-C7-ACP ester.…”

“…The strain FYJ5210 bearing pET28a:: bioH was grown to mid-log phase (OD600: ~0.8), and then subjected to 10h of induction with 0.5 mM IPTG at 16°C. The harvested bacterial cells were suspended with lysis buffer [20 mM Tris-HCl (pH8.0), 300 mM NaCl, 5% glycerol, 20 mM imidazole and 1 mM PMSF], and passed through 3 rounds of French Pressure Cell [ 62 ]. Following the removal of bacterial debris by the centrifugation at 16800 rpm for 1 h at 4°C, the resultant lysates were incubated with Ni-NTA agarose beads pre- equilibrated with lysis buffer for 1 h [ 17 , 54 ].…”

“…To visualize its metabolic role of P. aeruginosa BioH, we established the DTB/biotin bioassay coupled with the in vitro reconstituted system for DTB/biotin synthesis (Fig 4A and 4C) as described by different research groups [17,47,62]. In this BioH-including system, the cell-free crude extract was prepared from the biotin auxotroph of E. coli ΔbioH, providing an array of enzymes of FASII and late steps of biotin synthesis (Fig 4B).…”