Synthesis of the α,ω-dicarboxylic acid precursor of biotin by the canonical fatty acid biosynthetic pathway

Biotin synthesis requires the C7 α,ω-dicarboxylic acid, pimelic acid. Although pimelic acid was known to be primarily synthesized by a head to tail incorporation of acetate units, the synthetic mechanism was unknown. It has recently been demonstrated that in most bacteria the biotin pimelate moiety is synthesized by a modified fatty acid synthetic pathway in which the biotin synthetic intermediates are O-methyl esters disguised to resemble the canonical intermediates of the fatty acid synthetic pathway. Upon completion of the pimelate moiety, the methyl ester is cleaved. A very restricted set of bacteria have a different pathway in which the pimelate moiety is formed by cleavage of fatty acid synthetic intermediates by BioI, a member of the cytochrome P450 family.     

Identification and solution structures of a single domain biotin/lipoyl attachment protein from Bacillus subtilis

Protein biotinylation and lipoylation are post-translational modifications, in which biotin or lipoic acid is covalently attached to specific proteins containing biotin/lipoyl attachment domains. All the currently reported natural proteins containing biotin/lipoyl attachment domains are multidomain proteins and can only be modified by either biotin or lipoic acid in vivo. We have identified a single domain protein with 73 amino acid residues from Bacillus subtilis strain 168, and it can be both biotinylated and lipoylated in Escherichia coli. The protein is therefore named as biotin/lipoyl attachment protein (BLAP). This is the first report that a natural single domain protein exists as both a biotin and lipoic acid receptor. The solution structure of apo-BLAP showed that it adopts a typical fold of biotin/lipoyl attachment domain. The structure of biotinylated BLAP revealed that the biotin moiety is covalently attached to the side chain of Lys(35), and the bicyclic ring of biotin is folded back and immobilized on the protein surface. The biotin moiety immobilization is mainly due to an interaction between the biotin ureido ring and the indole ring of Trp(12). NMR study also indicated that the lipoyl group of the lipoylated BLAP is also immobilized on the protein surface in a similar fashion as the biotin moiety in the biotinylated protein.     

Contributions of the peroxisome and β-oxidation cycle to biotin synthesis in fungi

The first step in the synthesis of the bicyclic rings of D-biotin is mediated by 8-amino-7-oxononanoate (AON) synthase, which catalyzes the decarboxylative condensation of l-alanine and pimelate thioester. We found that the Aspergillus nidulans AON synthase, encoded by the bioF gene, is a peroxisomal enzyme with a type 1 peroxisomal targeting sequence (PTS1). Localization of AON to the peroxisome was essential for biotin synthesis because expression of a cytosolic AON variant or deletion of pexE, encoding the PTS1 receptor, rendered A. nidulans a biotin auxotroph. AON synthases with PTS1 are found throughout the fungal kingdom, in ascomycetes, basidiomycetes, and members of basal fungal lineages but not in representatives of the Saccharomyces species complex, including Saccharomyces cerevisiae. A. nidulans mutants defective in the peroxisomal acyl-CoA oxidase AoxA or the multifunctional protein FoxA showed a strong decrease in colonial growth rate in biotin-deficient medium, whereas partial growth recovery occurred with pimelic acid supplementation. These results indicate that pimeloyl-CoA is the in vivo substrate of AON synthase and that it is generated in the peroxisome via the β-oxidation cycle in A. nidulans and probably in a broad range of fungi. However, the β-oxidation cycle is not essential for biotin synthesis in S. cerevisiae or Escherichia coli. These results suggest that alternative pathways for synthesis of the pimelate intermediate exist in bacteria and eukaryotes and that Saccharomyces species use a pathway different from that used by the majority of fungi.     

The enzymes of biotin dependent CO2 metabolism: What structures reveal about their reaction mechanisms

Biotin is the major cofactor involved in carbon dioxide metabolism. Indeed, biotin‐dependent enzymes are ubiquitous in nature and are involved in a myriad of metabolic processes including fatty acid synthesis and gluconeogenesis. The cofactor, itself, is composed of a ureido ring, a tetrahydrothiophene ring, and a valeric acid side chain. It is the ureido ring that functions as the CO₂ carrier. A complete understanding of biotin‐dependent enzymes is critically important for translational research in light of the fact that some of these enzymes serve as targets for anti‐obesity agents, antibiotics, and herbicides. Prior to 1990, however, there was a dearth of information regarding the molecular architectures of biotin‐dependent enzymes. In recent years there has been an explosion in the number of three‐dimensional structures reported for these proteins. Here we review our current understanding of the structures and functions of biotin‐dependent enzymes. In addition, we provide a critical analysis of what these structures have and have not revealed about biotin‐dependent catalysis.     

Remarkable Diversity in the Enzymes Catalyzing the Last Step in Synthesis of the Pimelate Moiety of Biotin

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 activity against pimeloyl-ACP methyl ester, the physiological substrate of BioH. Two of the BioG proteins block biotin synthesis when highly expressed and these toxic proteins were shown to have more promiscuous substrate specificities than the non-toxic BioG proteins. A postulated BioG-BioC fusion protein was shown to functionally replace both the BioH and BioC functions of E. coli. Although the BioH, BioG and BioK esterases catalyze a common reaction, the proteins are evolutionarily distinct.     

Multi-level metabolic engineering of Pseudomonas mutabilis ATCC31014 for efficient production of biotin

Biotin (Vitamin H or B₇) is one of the most important cofactors involved in central metabolism of pro- and eukaryotic cells. Currently, chemical synthesis is the only route for commercial production. This study reports efficient microbial production of biotin in Pseudomonas mutabilis via multi-level metabolic engineering strategies: Level 1, overexpressing rate-limiting enzyme encoding genes involved in biotin synthesis (i.e. promoter and ribosome binding site engineering); Level 2, deregulating biotin biosynthesis (i.e. deletion of the negative regulator and the biotin importer genes); Level 3, enhancing the supply of co-factors (i.e. S-adenosyl-L-methionine and [Fe-S] cluster) for biotin biosynthesis; Level 4, increasing the availability of the precursor pimelate thioester (i.e. introduction of the BioW-BioI pathway from Bacillus subtilis). The combination of these interventions resulted in the establishment of a biotin overproducing strain, with the secretion of biotin increased for more than 460-fold. In combination with bioprocess engineering efforts, biotin was produced at a final titer of 87.17 mg/L in a shake flask and 271.88 mg/L in a fed-batch fermenter with glycerol as the carbon source. This is the highest biotin titer ever reported so far using rationally engineered microbial cell factories.     

Molecular dynamics investigations of BioH protein substrate specificity for biotin synthesis

BioH, an enzyme of biotin synthesis, plays an important role in fatty acid synthesis which assembles the pimelate moiety. Pimeloyl-acyl carrier protein (ACP) methyl ester, which is long known to be a biotin precursor, is the physiological substrate of BioH. Azelayl methyl ester, which has a longer chain than pimeloyl methyl ester, conjugated to ACP is also indeed accepted by BioH with very low rate of hydrolysis. To date, the substrate specificity for BioH and the molecular origin for the experimentally observed rate changes of hydrolysis by the chain elongation have remained elusive. To this end, we have investigated chain elongation effects on the structures by using the fully atomistic molecular dynamics simulations combined with binding free energy calculations. The results indicate that the substrate specificity is determined by BioH together with ACP. The added two methylenes would increase the structural flexibility by protein motions at the interface of ACP and BioH, instead of making steric clashes with the side chains of the BioH hydrophobic cavity. On the other hand, the slower hydrolysis of azelayl substrate is suggested to be associated with the loose of contacts between BioH and ACP, and with the lost electrostatic interactions of two ionic/hydrogen bonding networks at the interface of the two proteins. The present study provides important insights into the structure–function relationships of the complex of BioH with pimeloyl-ACP methyl ester, which could contribute to further understanding about the mechanism of the biotin synthetic pathway, including the catalytic role of BioH.     

Lipoic acid synthesis: a new family of octanoyltransferases generally annotated as lipoate protein ligases

Bacillus subtilis lacks a recognizable homologue of the LipB octanoyltransferase, an enzyme essential for lipoic acid synthesis in Escherichia coli. LipB transfers the octanoyl moiety from octanoyl-acyl carrier protein to the lipoyl domains of the 2-oxoacid dehydrogenases via a thioester-linked octanoyl-LipB intermediate. The octanoylated dehydrogenase is then converted to the enzymatically active lipoylated species by insertion of two sulfur atoms into the octanoyl moiety by the S-adenosyl-l-methionine radical enzyme, LipA (lipoate synthase). B. subtilis synthesizes lipoic acid and contains a LipA homologue that is fully functional in E. coli. Therefore, the lack of a LipB homologue presented the puzzle of how B. subtilis synthesizes the LipA substrate. We report that B. subtilis encodes an octanoyltransferase that has virtually no sequence resemblance to E. coli LipB but instead has a sequence that resembles that of the E. coli lipoate ligase, LplA. On the basis of this resemblance, these genes have generally been annotated as encoding a lipoate ligase, an enzyme that in E. coli scavenges lipoic acid from the environment but plays no role in de novo synthesis. We have named the B. subtilis octanoyltransferase LipM and find that, like LipB, the LipM reaction proceeds through a thioester-linked acyl enzyme intermediate. The LipM active site nucleophile was identified as C150 by the finding that this thiol becomes modified when LipM is expressed in E. coli. The level of the octanoyl-LipM intermediate can be significantly decreased by blocking fatty acid synthesis during LipM expression, and C150 was confirmed as an essential active site residue by site-directed mutagenesis. LipM homologues seem the sole type of octanoyltransferase present in the firmicutes and are also present in the cyanobacteria. LipM type octanoyltransferases represent a new clade of the PF03099 protein family, suggesting that octanoyl transfer activity has evolved at least twice within this superfamily.     

Expression, purification, and characterization of BioI: a carbon-carbon bond cleaving cytochrome P450 involved in biotin biosynthesis in Bacillus subtilis

Pimelic acid formation for biotin biosynthesis in Bacillus subtilis has been proposed to involve a cytochrome P450 encoded by the gene bioI. We have subcloned biol and overexpressed the encoded protein, Biol. A purification protocol was developed utilizing ion exchange, gel filtration, and hydroxyapatite chromatography. Investigation of the purified BioI by UV-visible spectroscopy revealed spectral properties characteristic of a cytochrome P450 enzyme. BioI copurifies with acylated Escherichia coli acyl carrier protein (ACP), suggesting that in vivo a fatty acid substrate may be presented to BioI as an acyl-ACP. A combination of electrospray mass spectrometry of the intact acyl-ACP and GCMS indicated a range of fatty acids were bound to the ACP. A catalytically active system has been established employing E. coli flavodoxin reductase and a novel, heterologous flavodoxin as the redox partners for BioI. In this system, BioI cleaves a carbon-carbon bond of an acyl-ACP to generate a pimeloyl-ACP equivalent, from which pimelic acid is isolated after base-catalyzed saponification. A range of free fatty acids have also been explored as potential alternative substrates for BioI, with C16 binding most tightly to the enzyme. These fatty acids are also metabolized to dicarboxylic acids, but with less regiospecificity than is observed with acyl-ACPs. A possible mechanism for this transformation is discussed. These results strongly support the proposed role for BioI in biotin biosynthesis. In addition, the production of pimeloyl-ACP explains the ability of BioI to function as a pimeloyl CoA source in E. coli, which, unlike B. subtilis, is unable to utilize free pimelic acid for biotin production.     

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.     

A bacterium that has three pathways to regulate membrane lipid fluidity

Well-studied bacteria such as Bacillus subtilis and Escherichia coli each have only a single pathway for synthesis of the unsaturated fatty acids required to make functional membrane lipids. In marked contrast, unsaturated fatty acid synthesis in Pseudomonas aeruginosa proceeds by three distinct pathways.     

RhlA converts beta-hydroxyacyl-acyl carrier protein intermediates in fatty acid synthesis to the beta-hydroxydecanoyl-beta-hydroxydecanoate component of rhamnolipids in Pseudomonas aeruginosa

Pseudomonas aeruginosa secretes a rhamnolipid (RL) surfactant that functions in hydrophobic nutrient uptake, swarming motility, and pathogenesis. We show that RhlA supplies the acyl moieties for RL biosynthesis by competing with the enzymes of the type II fatty acid synthase (FASII) cycle for the beta-hydroxyacyl-acyl carrier protein (ACP) pathway intermediates. Purified RhlA forms one molecule of beta-hydroxydecanoyl-beta-hydroxydecanoate from two molecules of beta-hydroxydecanoyl-ACP and is the only enzyme required to generate the lipid component of RL. The acyl groups in RL are primarily beta-hydroxydecanoyl, and in vitro, RhlA has a greater affinity for 10-carbon substrates, illustrating that RhlA functions as a molecular ruler that selectively extracts 10-carbon intermediates from FASII. Eliminating either FabA or FabI activity in P. aeruginosa increases RL production, illustrating that slowing down FASII allows RhlA to more-effectively compete for beta-hydroxydecanoyl-ACP. In Escherichia coli, the rate of fatty acid synthesis increases 1.3-fold when RhlA is expressed, to ensure the continued formation of fatty acids destined for membrane phospholipid even though 24% of the carbon entering FASII is diverted to RL synthesis. Previous studies have placed a ketoreductase, called RhlG, before RhlA in the RL biosynthetic pathway; however, our experiments show that RhlG has no role in RL biosynthesis. We conclude that RhlA is necessary and sufficient to form the acyl moiety of RL and that the flux of carbon through FASII accelerates to support RL production and maintain a supply of acyl chains for phospholipid synthesis.     

Multiple interactions between the transmembrane division proteins of Bacillus subtilis and the role of FtsL instability in divisome assembly

About 11 essential proteins assemble into a ring structure at the surface of the cell to bring about cytokinesis in bacteria. Several of these proteins have their major domains located outside the membrane, forming an assembly that we call the outer ring (OR). Previous work on division in Bacillus subtilis has shown that four of the OR proteins-FtsL, DivIC, DivIB, and PBP 2B-are interdependent for assembly. This contrasts with the mainly linear pathway for the equivalent proteins in Escherichia coli. Here we show that the interdependent nature of the B. subtilis pathway could be due to effects on FtsL and DivIC stability and that DivIB is an important player in regulating this turnover. Two-hybrid approaches suggest that a multiplicity of protein-protein interactions contribute to the assembly of the OR. DivIC is unusual in interacting strongly only with FtsL. We propose a model for the formation of the OR through the mutual association of the membrane proteins directed by the cytosolic inner-ring proteins.     

Cloning and characterization of biotin biosynthetic genes of Kurthia sp

The biotin biosynthesis genes of Kurthia sp., which is an aerobic gram-positive bacterium, were cloned from Kurthia sp. 538-KA26 and characterized. Eleven biotin biosynthetic genes have been identified in Kurthia sp. Kurthia sp. has two genes coding for KAPA synthase, bioF and bioFII, and also has two genes coding for BioH protein, bioH and bioHII. In addition, three genes, orf1, orf2, and orf3, whose functions are unknown, were found in the biotin gene clusters of Kurthia sp. The bioA, bioD, and orf1 genes are arranged in a gene cluster in the order orf1bioDA, and the bioB, bioF, and orf2 genes are arranged in a gene cluster in the order orf2bioFB. These gene clusters proceed to both directions; the face to face promoters and two 40-bp of palindrome sequences exist upstream of the orf1 and orf2 genes. The bioC, bioFII, and bioHII genes are arranged in a gene cluster in the order bioFIIHIIC; a 40-bp of palindrome sequence exists upstream of the bioFII gene. The bioH and orf3 genes are arranged in a gene cluster in the order bioHorf3; a palindrome sequence was not found upstream of the bioH gene. These palindrome sequences are extremely similar to each other, suggesting that the orf1bioDA, orf2bioFB, and bioFIIHIIC gene clusters are regulated by biotin. Kurthia sp. does not have the bioW gene coding pimeloyl-CoA synthase, suggesting that pimeloyl-CoA may be produced by a different pathway than that of gram-positive bacterium B. subtilis or B. sphaericus, further suggesting a modified fatty acid synthesis pathway via acetyl-CoA instead as E. coli has.     

NovJ/NovK catalyze benzylic oxidation of a beta-hydroxyl tyrosyl-S-pantetheinyl enzyme during aminocoumarin ring formation in novobiocin biosynthesis

The bicyclic coumarin ring in the aminocoumarin natural product antibiotics that target bacterial DNA gyrase is assembled from tyrosine by nonribosomal peptide synthetase logic. Tyrosine has previously been shown to be activated and installed as a phosphopantetheinyl thioester on the thiolation domain of NovH and then hydroxylated on the benzylic carbon by the heme protein NovI, generating beta-OH-Tyr-S-NovH. This aminoacyl-S-protein is the substrate for the next two orfs, Streptomyces sphaeroides NovJ and NovK, that have now been expressed in and purified from Escherichia coli as a J2K2 heterotetramer. NovJ/NovK use NADP as an electron acceptor to oxidize the beta-OH of the tyrosyl moiety to yield the tethered beta-ketotyrosyl-S-NovH. The enol tautomer is the form that predominates in the subsequently cyclized aminocoumarin scaffold. The labile beta-ketotyrosyl thioester moiety was identified by hydrolytic release from NovH, analysis by mass spectroscopy, and comparison with a synthetic sample. We also have identified a residue in NovJ that when mutated results in a 50-fold reduction in catalytic activity.     

Improved production of long-chain fatty acid in Escherichia coli by an engineering elongation cycle during fatty acid synthesis (FAS) through genetic manipulation

The microbial biosynthesis of fatty acid of lipid metabolism, which can be used as precursors for the production of fuels of chemicals from renewable carbon sources, has attracted significant attention in recent years. The regulation of fatty acid biosynthesis pathways has been mainly studied in a model prokaryote, Escherichia coli. During the recent period, global regulation of fatty acid metabolic pathways has been demonstrated in another model prokaryote, Bacillus subtilis, as well as in Streptococcus pneumonia. The goal of this study was to increase the production of long-chain fatty acids by developing recombinant E. coli strains that were improved by an elongation cycle of fatty acid synthesis (FAS). The fabB, fabG, fabZ, and fabI genes, all homologous of E. coli, were induced to improve the enzymatic activities for the purpose of overexpressing components of the elongation cycle in the FAS pathway through metabolic engineering. The beta-oxoacyl-ACP synthase enzyme catalyzed the addition of acyl-ACP to malonyl-ACP to generate beta- oxoacyl-ACP. The enzyme encoded by the fabG gene converted beta-oxoacyl-ACP to beta-hydroxyacyl-ACP, the fabZ catalyzed the dehydration of beta-3-hydroxyacyl-ACP to trans-2-acyl-ACP, and the fabI gene converted trans-2- acyl-ACP to acyl-ACP for long-chain fatty acids. In vivo productivity of total lipids and fatty acids was analyzed to confirm the changes and effects of the inserted genes in E. coli. As a result, lipid was increased 2.16-fold higher and hexadecanoic acid was produced 2.77-fold higher in E. coli JES1030, one of the developed recombinants through this study, than those from the wild-type E. coli.     

Biosynthesis of 5-alkylresorcinol in rice: incorporation of a putative fatty acid unit in the 5-alkylresorcinol carbon chain

A previously unspecified "starter" unit in the predicted biosynthesis pathway of 5-alkylresorcinols has now identified as a fatty acid or its equivalent, using an efficient 5-alkylresorcinol production system of etiolated rice seedlings. Feeding saturated, odd-carbon fatty acid ester substrates from C11 to C19 specifically and markedly increased the amount of the corresponding 5-alkylresorcinol homologs with even-carbon chains that are shorter by one carbon than those of the supplied fatty acids. The amount of these homologs depended on substrate concentration. Some of the homologs whose amounts increased had linear carbon chains and the dodecyl homolog was shown to be 5-n-dodecylresorcinol. Moreover, the 13C label in the dodecyl homolog that was biosynthesized from the [1-13C]tridecanoate substrate was localized on the C-5 carbon of the resorcinol ring. These results obviously show that the fatty acid unit acts as a direct precursor and forms the side-chain moiety of 5-n-alkylresorcinol via the predicted biosynthesis pathway.     

Rapid functional identification of putative genes based on the combined in vitro protein synthesis with mass spectrometry: a tool for functional genomics

For the rapid identification of functional activity of unknown genes from a sequence database, a new method based on in vitro protein synthesis combined with mass spectrometry was developed. To discriminate their subtle enzymatic activity, in vitro synthesized and one-step purified lipolytic enzymes, such as lip A and lip B from Bacillus subtilis and an unknown protein ybfF from Escherichia coli, were reacted with a mixture of triglycerides with different carbon chain lengths. Using direct matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of reaction product, all three enzymes were revealed to have strong esterase activity rather than true lipase activity, which has no reactivity on long-chain fatty acids such as triolein. These results were also confirmed by classical color assay using p-nitrophenyl butyrate (pNPB) and p-nitrophenyl palmitate (pNPP) as representative lipolytic substrates.     

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.     

Kinetic characterization of mutations found in propionic acidemia and methylcrotonylglycinuria: evidence for cooperativity in biotin carboxylase

Acetyl-CoA carboxylase catalyzes the committed step in fatty acid synthesis in all plants, animals, and bacteria. The Escherichia coli form is a multifunctional enzyme consisting of three separate proteins: biotin carboxylase, carboxyltransferase, and the biotin carboxyl carrier protein. The biotin carboxylase component, which catalyzes the ATP-dependent carboxylation of biotin using bicarbonate as the carboxylate source, has a homologous functionally identical subunit in the mammalian biotin-dependent enzymes propionyl-CoA carboxylase and 3-methylcrotonyl-CoA carboxylase. In humans, mutations in either of these enzymes result in the metabolic deficiency propionic acidemia or methylcrotonylglycinuria. The lack of a system for structure-function studies of these two biotin-dependent carboxylases has prevented a detailed analysis of the disease-causing mutations. However, structural data are available for E. coli biotin carboxylase as is a system for its overexpression and purification. Thus, we have constructed three site-directed mutants of biotin carboxylase that are homologous to three missense mutations found in propionic acidemia or methylcrotonylglycinuria patients. The mutants M169K, R338Q, and R338S of E. coli biotin carboxylase were selected for study to mimic the disease-causing mutations M204K and R374Q of propionyl-CoA carboxylase and R385S of 3-methylcrotonyl-CoA carboxylase. These three mutants were subjected to a rigorous kinetic analysis to determine the function of the residues in the catalytic mechanism of biotin carboxylase as well as to establish a molecular basis for the two diseases. The results of the kinetic studies have revealed the first evidence for negative cooperativity with respect to bicarbonate and suggest that Arg-338 serves to orient the carboxyphosphate intermediate for optimal carboxylation of biotin.