Abstract:Biotin synthesis in Escherichia coli requires the functions of the bioH and bioC genes to synthesize the precursor pimelate moiety by use of a modified fatty acid biosynthesis pathway. However, it was previously noted that bioH has been replaced with bioG or bioK within the biotin synthetic gene clusters of other bacteria. We report that each of four BioG proteins from diverse bacteria and two cyanobacterial BioK proteins functionally replace E. coli BioH in vivo. Moreover, purified BioG proteins have esterase… Show more
“…2c). Note that despite its low sequence identity with E. coli BioH the P. aeruginosa protein is clearly a BioH and not one of the other pimeloyl-ACP methyl ester esterases because those nonorthologous proteins cannot be aligned with E. coli BioH even given very permissive alignment parameters 24, 25, 30 .Figure 2Sequence alignments, purification and modeling of BioH proteins. (A), sequence alignment of the BioH proteins of E. coli and P. aeruginosa PAO1.…”
BioH is an α/β-hydrolase required for synthesis of the pimelate moiety of biotin in diverse bacteria. The bioH gene is found in different genomic contexts. In some cases (e.g., Escherichia coli) the gene is not located within a biotin synthetic operon and its transcription is not coregulated with the other biotin synthesis genes. In other genomes such as Pseudomonas aeruginosa the bioH gene is within a biotin synthesis operon and its transcription is coregulated with the other biotin operon genes. The esterases of pimelate moiety synthesis show remarkable genomic plasticity in that in some biotin operons bioH is replaced by other α/ß hydrolases of diverse sequence. The “wild card” nature of these enzymes led us to compare the paradigm “freestanding” E. coli BioH with the operon-encoded P. aeruginosa BioH. We hypothesized that the operon-encoded BioH might differ in its expression level and/or activity from the freestanding BioH gene. We report this is not the case. The two BioH proteins show remarkably similar hydrolase activities and substrate specificity. Moreover, Pseudomonas aeruginosa BioH is more highly expressed than E. coli BioH. Despite the enzymatic similarities of the two BioH proteins, bioinformatics analysis places the freestanding and operon-encoded BioH proteins into distinct clades.
“…2c). Note that despite its low sequence identity with E. coli BioH the P. aeruginosa protein is clearly a BioH and not one of the other pimeloyl-ACP methyl ester esterases because those nonorthologous proteins cannot be aligned with E. coli BioH even given very permissive alignment parameters 24, 25, 30 .Figure 2Sequence alignments, purification and modeling of BioH proteins. (A), sequence alignment of the BioH proteins of E. coli and P. aeruginosa PAO1.…”
BioH is an α/β-hydrolase required for synthesis of the pimelate moiety of biotin in diverse bacteria. The bioH gene is found in different genomic contexts. In some cases (e.g., Escherichia coli) the gene is not located within a biotin synthetic operon and its transcription is not coregulated with the other biotin synthesis genes. In other genomes such as Pseudomonas aeruginosa the bioH gene is within a biotin synthesis operon and its transcription is coregulated with the other biotin operon genes. The esterases of pimelate moiety synthesis show remarkable genomic plasticity in that in some biotin operons bioH is replaced by other α/ß hydrolases of diverse sequence. The “wild card” nature of these enzymes led us to compare the paradigm “freestanding” E. coli BioH with the operon-encoded P. aeruginosa BioH. We hypothesized that the operon-encoded BioH might differ in its expression level and/or activity from the freestanding BioH gene. We report this is not the case. The two BioH proteins show remarkably similar hydrolase activities and substrate specificity. Moreover, Pseudomonas aeruginosa BioH is more highly expressed than E. coli BioH. Despite the enzymatic similarities of the two BioH proteins, bioinformatics analysis places the freestanding and operon-encoded BioH proteins into distinct clades.
“…The biochemical pathway for biotin synthesis in analogy to the pathway in Escherichia coli is expected to proceed by methylation of malonyl-ACP 1 to the corresponding methyl ester 2 by BioC, which allows it to enter the fatty acid synthesis pathway (FAS-II) where it undergo two rounds of extension to pimeloyl-ACP methyl ester 3 (Lin, et al, 2010; Shapiro, et al, 2012). Hydrolysis of the methyl ester 3 by BioH is expected to furnish pimeloyl-ACP 4 .…”
SUMMARY
Biotin biosynthesis is essential for survival and persistence of Mycobacterium tuberculosis (Mtb) in vivo. The aminotransferase BioA, which catalyzes the antepenultimate step in the biotin pathway, has been established as a promising target due to its vulnerability to chemical inhibition. We performed high-throughput screening (HTS) employing a fluorescence displacement assay and identified a diverse set of potent inhibitors including many diversity-oriented synthesis (DOS) scaffolds. To efficiently select only hits targeting biotin biosynthesis, we then deployed a whole-cell counter-screen in either biotin-free and biotin-containing medium against wild-type Mtb and in parallel with isogenic bioA Mtb strains that possess differential levels of BioA expression. This counter-screen proved crucial to filter out compounds whose whole-cell activity was off-target as well as identify hits with weak, but measurable whole-cell activity in BioA-depleted strains. Several of the most promising hits were co-crystallized with BioA to provide a framework for future structure-based drug design efforts.
“…Thus, the E. coli bioH gene may encode a protein that is less specific than those encoded by the more “domesticated” bioH genes. Note that the BioH function seems something of a “wild card” among biotin synthetic enzymes since in some bacteria the gene has been displaced from its site upstream of bioC by other genes (79) that have been shown functionally replace BioH (81). …”
Section: Remaining Problems In Biotin Synthesismentioning
Summary
Two vitamins, biotin and lipoic acid, are essential in all three domains of life. Both coenzymes function only when covalently attached to key metabolic enzymes. There they act as “swinging arms” that shuttle intermediates between two active sites (= covalent substrate channeling) of key metabolic enzymes. Although biotin was discovered over 100 years ago and lipoic acid 60 years ago, it was not known how either coenzyme is made until recently. In Escherichia coli the synthetic pathways for both coenzymes have now been worked out for the first time.
The late steps of biotin synthesis, those involved in assembling the fused rings, were well-described biochemically years ago, although recent progress has been made on the BioB reaction, the last step of the pathway in which the biotin sulfur moiety is inserted. In contrast, the early steps of biotin synthesis, assembly of the fatty acid-like “arm” of biotin were unknown. It has now been demonstrated that the arm is made by using disguised substrates to gain entry into the fatty acid synthesis pathway followed by removal of the disguise when the proper chain length is attained. The BioC methyltransferase is responsible for introducing the disguise and the BioH esterase for its removal.
In contrast to biotin, which is attached to its cognate proteins as a finished molecule, lipoic acid is assembled on its cognate proteins. An octanoyl moiety is transferred from the octanoyl-ACP of fatty acid synthesis to a specific lysine residue of a cognate protein by the LipB octanoyl transferase followed by sulfur insertion at carbons C6 and C8 by the LipA lipoyl synthetase. Assembly on the cognate proteins regulates the amount of lipoic acid synthesized and thus there is no transcriptional control of the synthetic genes. In contrast transcriptional control of the biotin synthetic genes is wielded by a remarkably sophisticated, yet simple, system, exerted through BirA a dual function protein that both represses biotin operon transcription and ligates biotin to its cognate protein.
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