Biosynthesis of biotin: synthesis of 7,8-diaminopelargonic acid in cell-free extracts of Escherichia coli

Cell-free extracts prepared from a biotin auxotroph of Escherichia coli were active in catalyzing the synthesis of 7,8-diaminopelargonic acid, an intermediate of the biotin pathway, from 7-oxo-8-aminopelargonic acid. The product was identified on the basis of its chromatographic characteristics and its biotin activities for biotin auxotrophs of E. coli. Enzyme activity was determined in a reaction coupled with the desthiobiotin synthetase system, which is required for the conversion of 7,8-diaminopelargonic acid to desthiobiotin, and by measuring the amount of desthiobiotin formed by microbiological assay. The reaction was stimulated by l-methionine and pyridoxal-5'-phosphate. l-Methionine could not be replaced by any other amino acids tested. Pyridoxamine and pyridoxamine-5'-phosphate were as active as pyridoxal phosphate. The enzyme, presumably an aminotransferase, was demonstrable in the parent strain of E. coli and all mutant strains tested with the exception of a strain which is able to grow on diaminopelargonic acid but not on 7-oxo-8-aminopelargonic acid. Furthermore, the enzyme was repressible by biotin. The results were consistent with the hypothesis that the biosynthesis of 7,8-diaminopelargonic acid from 7-oxo-8-aminopelargonic acid is an obligatory step in the biosynthetic pathway of biotin in E. coli.     

The C-terminal domain of biotin protein ligase from E. coli is required for catalytic activity

Biotin protein ligase of Escherichia coli, the BirA protein, catalyses the covalent attachment of the biotin prosthetic group to a specific lysine of the biotin carboxyl carrier protein (BCCP) subunit of acetyl-CoA carboxylase. BirA also functions to repress the biotin biosynthetic operon and synthesizes its own corepressor, biotinyl-5'-AMP, the catalytic intermediate in the biotinylation reaction. We have previously identified two charge substitution mutants in BCCP, E119K, and E147K that are poorly biotinylated by BirA. Here we used site-directed mutagenesis to investigate residues in BirA that may interact with E119 or E147 in BCCP. None of the complementary charge substitution mutations at selected residues in BirA restored activity to wild-type levels when assayed with our BCCP mutant substrates. However, a BirA variant, in which K277 of the C-terminal domain was substituted with Glu, had significantly higher activity with E119K BCCP than did wild-type BirA. No function has been identified previously for the BirA C-terminal domain, which is distinct from the central domain thought to contain the ATP binding site and is known to contain the biotin binding site. Kinetic analysis of several purified mutant enzymes indicated that a single amino acid substitution within the C-terminal domain (R317E) and located some distance from the presumptive ATP binding site resulted in a 25-fold decrease in the affinity for ATP. Our data indicate that the C-terminal domain of BirA is essential for the catalytic activity of the enzyme and contributes to the interaction with ATP and the protein substrate, the BCCP biotin domain.     

Complementation of anArabidopsis thaliana biotin auxotroph with anEscherichia coli biotin biosynthetic gene

TheArabidopsis thaliana biotin auxotrophbio1 was rendered prototrophic by transformation with a chimeric transgene containing theEscherichia coli bioA gene driven by a constitutive promoter. ThebioA gene encodes the biotin biosynthetic enzyme 7,8-diaminopelargonic acid aminotransferase. Unlike the untransformed control plants, transgenic plants expressing the bacterial transgene synthesized biotin and grew to maturity without biotin-deficiency symptoms. These findings demonstrate thatbio1/bio1 mutant plants are defective in the gene encoding 7,8-diaminopelargonic acid aminotransferase.     

Complementation of anArabidopsis thaliana biotin auxotroph with anEscherichia coli biotin biosynthetic gene

TheArabidopsis thaliana biotin auxotrophbio1 was rendered prototrophic by transformation with a chimeric transgene containing theEscherichia coli bioA gene driven by a constitutive promoter. ThebioA gene encodes the biotin biosynthetic enzyme 7,8-diaminopelargonic acid aminotransferase. Unlike the untransformed control plants, transgenic plants expressing the bacterial transgene synthesized biotin and grew to maturity without biotin-deficiency symptoms. These findings demonstrate thatbio1/bio1 mutant plants are defective in the gene encoding 7,8-diaminopelargonic acid aminotransferase.     

Biosynthesis of Desthiobiotin in Cell-free Extracts of Escherichia coli

Cell-free extracts of Escherichia coli were active in catalyzing the synthesis of a biotin vitamer from 7,8-diaminopelargonic acid. The vitamer was identified as desthiobiotin on the basis of its chromatographic and electrophoretic characteristics and its biotin activities for a variety of microorganisms. The reaction was stimulated five-fold by bicarbonate, suggesting that an "active CO(2)" was incorporated into the carbonyl carbon of desthiobiotin. The enzyme was demonstrable in a wild-type (K-12) and in all biotin mutants of E. coli that were tested, with the exception of a strain which was able to grow on desthiobiotin but not on diaminopelargonic acid. Furthermore, the enzyme was repressible by biotin in all of the strains tested. These results are consistent with the hypothesis that the biosynthesis of desthiobiotin from 7,8-diaminopelargonic acid is an obligatory step in the biosynthetic pathway of biotin in E. coli.     

MMAR_2770, a new enzyme involved in biotin biosynthesis, is essential for the growth of Mycobacterium marinum in macrophages and zebrafish

Biotin, which functions as an essential cofactor for certain carboxylases and decarboxylases, is synthesized by a multistep pathway in microorganisms and plants. Biotin biosynthesis has not been studied in detail in mycobacteria. In this study, we isolated a mutant of Mycobacterium marinum in which MMAR_2770, a previously uncharacterized gene encoding a predicted short-chain dehydrogenase/reductase, was inactivated. We found that this mutant is a biotin auxotroph that cannot grow in a minimal medium (Sauton) unless biotin is supplemented. Complementation of the mutant with an intact MMAR_2770 or its homolog Rv1882c of Mycobacterium tuberculosis restored the growth of the mutant, suggesting that MMAR_2770 is involved in biotin biosynthesis. We further showed that the mutant was unable to grow in cultured macrophages and was attenuated in zebrafish. Taken together, our results demonstrate that biotin biosynthesis is essential for the growth of mycobacteria in vitro and in vivo and have provided validation for targeting biotin biosynthetic enzymes for antimycobacterial drug development. The potential role of MMAR_2770 in mycobacterial biotin biosynthesis is discussed.     

Biotin synthesis in plants. The first committed step of the pathway is catalyzed by a cytosolic 7-keto-8-aminopelargonic acid synthase

Biochemical and molecular characterization of the biotin biosynthetic pathway in plants has dealt primarily with biotin synthase. This enzyme catalyzing the last step of the pathway is localized in mitochondria. Other enzymes of the pathway are however largely unknown. In this study, a genomic-based approach allowed us to clone an Arabidopsis (Arabidopsis thaliana) cDNA coding 7-keto-8-aminopelargonic acid (KAPA) synthase, the first committed enzyme of the biotin synthesis pathway, which we named AtbioF. The function of the enzyme was demonstrated by functional complementation of an Escherichia coli mutant deficient in KAPA synthase reaction, and by measuring in vitro activity. Overproduction and purification of recombinant AtbioF protein enabled a thorough characterization of the kinetic properties of the enzyme and a spectroscopic study of the enzyme interaction with its substrates and product. This is the first characterization of a KAPA synthase reaction in eukaryotes. Finally, both green fluorescent protein-targeting experiments and western-blot analyses showed that the Arabidopsis KAPA synthase is present in cytosol, thus revealing a unique compartmentation of the plant biotin synthesis, split between cytosol and mitochondria. The significance of the complex compartmentation of biotin synthesis and utilization in the plant cell and its potential importance in the regulation of biotin metabolism are also discussed.     

Mutations at four active site residues of biotin carboxylase abolish substrate-induced synergism by biotin

Acetyl-CoA carboxylase catalyzes the first committed step in the biosynthesis of long-chain fatty acids. The Escherichia coli form of the enzyme consists of a biotin carboxylase protein, a biotin carboxyl carrier protein, and a carboxyltransferase protein. In this report a system for site-directed mutagenesis of the biotin carboxylase component is described. The wild-type copy of the enzyme, derived from the chromosomal gene, is separated from the mutant form of the enzyme which is coded on a plasmid. Separation of the two forms is accomplished using a histidine-tag attached to the amino terminus of the mutant form of the enzyme and nickel affinity chromatography. This system was used to mutate four active site residues, E211, E288, N290, and R292, to alanine followed by their characterization with respect to several different reactions catalyzed by biotin carboxylase. In comparison to wild-type biotin carboxylase, all four mutant enzymes gave very similar results in all the different assays, suggesting that the mutated residues have a common function. The mutations did not affect the bicarbonate-dependent ATPase reaction. In contrast, the mutations decreased the maximal velocity of the biotin-dependent ATPase reaction 1000-fold but did not affect the Km for biotin. The activity of the ATP synthesis reaction catalyzed by biotin carboxylase where carbamoyl phosphate reacts with ADP was decreased 100-fold by the mutations. The ATP synthesis reaction required biotin to stimulate the activity in the wild-type; however, biotin did not stimulate the activity of the mutant enzymes. The results showed that the mutations have abolished the ability of biotin to increase the activity of the enzyme. Thus, E211, E288, N290, and R292 were responsible, at least in part, for the substrate-induced synergism by biotin in biotin carboxylase.     

The gene encoding the biotin carboxylase subunit of Escherichia coli acetyl-CoA carboxylase.

We report the molecular cloning and DNA sequence of the gene encoding the biotin carboxylase subunit of Escherichia coli acetyl-CoA carboxylase. The biotin carboxylase gene encodes a protein of 449 residues that is strikingly similar to amino-terminal segments of two biotin-dependent carboxylase proteins, yeast pyruvate carboxylase and the alpha-subunit of rat propionyl-CoA carboxylase. The deduced biotin carboxylase sequence contains a consensus ATP binding site and a cysteine-containing sequence preserved in all sequenced bicarbonate-dependent biotin carboxylases that may play a key catalytic role. The gene encoding the biotin carboxyl carrier protein (BCCP) subunit of acetyl-CoA carboxylase is located upstream of the biotin carboxylase gene and the two genes are cotranscribed. As previously reported by others, the BCCP sequence encoded a protein of 16,688 molecular mass. However, this value is much smaller than that (22,500 daltons) obtained by analysis of the protein. Amino-terminal amino acid sequencing of the purified BCCP protein confirmed the deduced amino acid sequence indicating that BCCP is a protein of atypical physical properties. Northern and primer extension analyses demonstrate that BCCP and biotin carboxylase are transcribed as a single mRNA species that contains an unusually long untranslated leader preceding the BCCP gene. We have also determined the mutational alteration in a previously isolated acetyl-CoA carboxylase (fabE) mutant and show the lesion maps within the BCCP gene and results in a BCCP species defective in acceptance of biotin. Translational fusions of the carboxyl-terminal 110 or 84 (but not 76) amino acids of BCCP to beta-galactosidase resulted in biotinated beta-galactosidase molecules and production of one such fusion was shown to result in derepression of the biotin biosynthetic operon.     

Synthesis of 7-oxo-8-aminopelargonic acid, a biotin vitamer, in cell-free extracts of Escherichia coli biotin auxotrophs

The enzymatic synthesis of 7-oxo-8-aminopelargonic acid (7-KAP) from pimelyl-coenzyme A and l-alanine was demonstrated in cell-free extracts of a biotin mutant of Escherichia coli K-12 which excretes only 7-KAP into the growth medium. This biotin vitamer was identified by its chromatographic and electrophoretic properties. The enzyme (7-KAP synthetase) was repressed when the organism was grown in biotin concentrations greater than 0.2 ng/ml. The parent strain and members of other mutant groups that excrete 7-KAP, in addition to other vitamers, also exhibited synthetase activity. A mutant group that failed to excrete 7-KAP was further sub-divided into three groups, one of which lacked synthetase activity. These results are discussed in relation to a previously proposed scheme for biotin biosynthesis in which the formation of 7-KAP is considered the point of entry for pimelic acid into the biotin pathway.     

Identification and characterization of a new enoyl coenzyme A hydratase involved in biosynthesis of medium-chain-length polyhydroxyalkanoates in recombinant Escherichia coli

The biosynthetic pathway of medium-chain-length (MCL) polyhydroxyalkanoates (PHAs) from fatty acids has been established in fadB mutant Escherichia coli strain by expressing the MCL-PHA synthase gene. However, the enzymes that are responsible for the generation of (R)-3-hydroxyacyl coenzyme A (R3HA-CoAs), the substrates for PHA synthase, have not been thoroughly elucidated. Escherichia coli MaoC, which is homologous to Pseudomonas aeruginosa (R)-specific enoyl-CoA hydratase (PhaJ1), was identified and found to be important for PHA biosynthesis in a fadB mutant E. coli strain. When the MCL-PHA synthase gene was introduced, the fadB maoC double-mutant E. coli WB108, which is a derivative of E. coli W3110, accumulated 43% less amount of MCL-PHA from fatty acid compared with the fadB mutant E. coli WB101. The PHA biosynthetic capacity could be restored by plasmid-based expression of the maoCEc gene in E. coli WB108. Also, E. coli W3110 possessing fully functional beta-oxidation pathway could produce MCL-PHA from fatty acid by the coexpression of the maoCEc gene and the MCL-PHA synthase gene. For the enzymatic analysis, MaoC fused with His6-Tag at its C-terminal was expressed in E. coli and purified. Enzymatic analysis of tagged MaoC showed that MaoC has enoyl-CoA hydratase activity toward crotonyl-CoA. These results suggest that MaoC is a new enoyl-CoA hydratase involved in supplying (R)-3-hydroxyacyl-CoA from the beta-oxidation pathway to PHA biosynthetic pathway in the fadB mutant E. coli strain.     

On the mechanism of conversion of dethiobiotin to biotin in Escherichia coli. Discussion of the occurrence of an intermediate hydroxylation.

The last step of the biosynthesis of biotin, i.e. the conversion of dethiobiotin to biotin was studied using E. coli. The three dethiobiotin derivatives hydroxylated at C-2 or C-5 were synthesized and tested as potential precursors of biotin. It appears that none of these compounds is able to support the growth of E. coli C124, a mutant which does not synthesize dethiobiotin, but converts it into biotin. These results strongly disfavour the hypothesis of the activation of the saturated carbons by an hydroxylation process.     

A high-throughput screening assay for the carboxyltransferase subunit of acetyl-CoA carboxylase

One consequence of the dramatic rise of antibiotic-resistant pathogenic bacteria is the need for new targets for antibiotics. Because membrane lipid biogenesis is essential for bacterial growth, enzymes of the fatty acid biosynthetic pathway offer attractive possibilities for the development of new antibiotics. Acetyl-coenzyme A carboxylase (ACC) catalyzes the first committed and regulated step in fatty acid biosynthesis in bacteria and thus is a prime target for development of antibiotics. ACC is a multifunctional enzyme composed of three separate proteins. The biotin carboxylase component catalyzes the ATP-dependent carboxylation of biotin. The biotin carboxyl carrier protein features a biotin molecule covalently attached at Lys122 of the Escherichia coli enzyme. The carboxyltransferase subunit catalyzes the transfer of a carboxyl group from biotin to acetyl-coenzyme A (acetyl-CoA) to form malonyl-CoA. The objective of this study was to develop an assay for high-throughput screening for inhibitors of the carboxyltransferase subunit. The carboxyltransferase reaction was assayed in the reverse direction in which malonyl-CoA reacts with biocytin (an analog of the biotin carboxyl carrier protein) to form acetyl-CoA and carboxybiotin. The production of acetyl-CoA was coupled to citrate synthase, which produced citrate and coenzyme A. The amount of coenzyme A formed was detected using 5,5'-dithiobis(2-nitrobenzoic acid) (Ellman's reagent). The assay has been developed for use in both 96- and 384-well microplate formats and was validated using a known bisubstrate analog inhibitor of carboxyltransferase. The spectrophotometric readout in the visible absorbance range used in this assay does not generate the number of false negatives associated with frequently used NAD/NADH assay systems that rely on detection of NADH using UV absorbance.     

Targeted and proximity-dependent promiscuous protein biotinylation by a mutant Escherichia coli biotin protein ligase

A method for general protein biotinylation by enzymatic means has been developed. A mutant form (R118G) of the biotin protein ligase (BirA) of Escherichia coli is used and biotinylation is thought to proceed by chemical acylation of protein lysine side chains by biotinoyl-5'-AMP released from the mutant protein. Bovine serum albumin, chloramphenicol acetyltransferase, immunoglobulin chains and RNAse A as well as a large number of E. coli proteins have been biotinylated. The biotinylation reaction is proximity dependent in that the extent of biotinylation is much greater when the ligase is coupled to the acceptor protein than when the acceptor is free in solution. This is presumably due to rapid hydrolysis of the acylation agent, biotinoyl-5'-AMP. Therefore, when the mutant ligase is attached to one partner involved in a protein-protein interaction, it can be used to specifically tag the other partner with biotin, thereby permitting facile detection and recovery of the proteins by existing avidin/streptavidin technology.     

An Embryo-Defective Mutant of Arabidopsis Disrupted in the Final Step of Biotin Synthesis

Auxotrophic mutants have played an important role in the genetic dissection of biosynthetic pathways in microorganisms. Equivalent mutants have been more difficult to identify in plants. The bio1 auxotroph of Arabidopsis thaliana was shown previously to be defective in the synthesis of the biotin precursor 7,8-diaminopelargonic acid. A second biotin auxotroph of A. thaliana has now been identified. Arrested embryos from this bio2 mutant are defective in the final step of biotin synthesis, the conversion of dethiobiotin to biotin. This enzymatic reaction, catalyzed by the bioB product (biotin synthase) in Escherichia coli, has been studied extensively in plants and bacteria because it involves the unusual addition of sulfur to form a thiophene ring. Three lines of evidence indicate that bio2 is defective in biotin synthase production: mutant embryos are rescued by biotin but not dethiobiotin, the mutant allele maps to the same chromosomal location as the cloned biotin synthase gene, and gel-blot hybridizations and polymerase chain reaction amplifications revealed that homozygous mutant plants contain a deletion spanning the entire BIO2-coding region. Here we describe how the isolation and characterization of this null allele have provided valuable insights into biotin synthesis, auxotrophy, and gene redundancy in plants.     

Cloning and characterization of the Bacillus subtilis birA gene encoding a repressor of the biotin operon.

The Bacillus subtilis birA gene, which regulates biotin biosynthesis, has been cloned and characterized. The birA gene maps at 202 degrees on the B. subtilis chromosome and encodes a 36,200-Da protein that is 27% identical to Escherichia coli BirA protein. Three independent mutations in birA that lead to deregulation of biotin synthesis alter single amino acids in the amino-terminal end of the protein. The amino-terminal region that is affected by these three birA mutations shows sequence similarity to the helix-turn-helix DNA binding motif previously identified in E. coli BirA protein. B. subtilis BirA protein also possesses biotin-protein ligase activity, as judged by its ability to complement a conditional lethal birA mutant of E. coli.     

Closing in on complete pathways of biotin biosynthesis

Biotin is an enzyme cofactor indispensable to metabolic fixation of carbon dioxide in all three domains of life. Although the catalytic and physiological roles of biotin have been well characterized, the biosynthesis of biotin remains to be fully elucidated. Studies in microbes suggest a two-stage biosynthetic pathway in which a pimelate moiety is synthesized and used to begin assembly of the biotin bicyclic ring structure. The enzymes involved in the bicyclic ring assembly have been studied extensively. In contrast the synthesis of pimelate, a seven carbon α,ω-dicarboxylate, has long been an enigma. Support for two different routes of pimelate synthesis has recently been obtained in Escherichia coli and Bacillus subtilis. The E. coli BioC-BioH pathway employs a methylation and demethylation strategy to allow elongation of a temporarily disguised malonate moiety to a pimelate moiety by the fatty acid synthetic enzymes whereas the B. subtilis BioI-BioW pathway utilizes oxidative cleavage of fatty acyl chains. Both pathways produce the pimelate thioester precursor essential for the first step in assembly of the fused rings of biotin. The enzymatic mechanisms and biochemical strategies of these pimelate synthesis models will be discussed in this review.     

Transfer of the high-GC cyclohexane carboxylate degradation pathway from Rhodopseudomonas palustris to Escherichia coli for production of biotin

This work demonstrates the transfer of the five-gene cyclohexane carboxylate (CHC) degradation pathway from the high-GC alphaproteobacterium Rhodopseudomonas palustris to Escherichia coli, a gammaproteobacterium. The degradation product of this pathway is pimeloyl-CoA, a key metabolite in E. coli's biotin biosynthetic pathway. This pathway is useful for biotin overproduction in E. coli; however, the expression of GC-rich genes is troublesome in this host. When the native R. palustris CHC degradation pathway is transferred to a DeltabioH pimeloyl-CoA auxotroph of E. coli, it is unable to complement growth in the presence of CHC. To overcome this expression problem we redesigned the operon with decreased GC content and removed stretches of high-GC intergenic DNA which comprise the 5' untranslated region of each gene, replacing these features with shorter low-GC sequences. We show this synthetic construct enables growth of the DeltabioH strain in the presence of CHC. When the synthetic degradation pathway is overexpressed in conjunction with the downstream genes for biotin biosynthesis, we measured significant accumulation of biotin in the growth medium, showing that the pathway transfer is successfully integrated with the host metabolism.