Structure and substrate selectivity of the 750-kDa ?6?6 holoenzyme of geranyl-CoA carboxylase.
ABSTRACT: Geranyl-CoA carboxylase (GCC) is essential for the growth of Pseudomonas organisms with geranic acid as the sole carbon source. GCC has the same domain organization and shares strong sequence conservation with the related biotin-dependent carboxylases 3-methylcrotonyl-CoA carboxylase (MCC) and propionyl-CoA carboxylase (PCC). Here we report the crystal structure of the 750-kDa ?6?6 holoenzyme of GCC, which is similar to MCC but strikingly different from PCC. The structures provide evidence in support of two distinct lineages of biotin-dependent acyl-CoA carboxylases, one carboxylating the ? carbon of a saturated organic acid and the other carboxylating the ? carbon of an ?-? unsaturated acid. Structural differences in the active site region of GCC and MCC explain their distinct substrate preferences. Especially, a glycine residue in GCC is replaced by phenylalanine in MCC, which blocks access by the larger geranyl-CoA substrate. Mutation of this residue in the two enzymes can change their substrate preferences.
Project description:3-Methylcrotonyl-CoA carboxylase (MCC), a member of the biotin-dependent carboxylase superfamily, is essential for the metabolism of leucine, and deficient mutations in this enzyme are linked to methylcrotonylglycinuria (MCG) and other serious diseases in humans. MCC has strong sequence conservation with propionyl-CoA carboxylase (PCC), and their holoenzymes are both 750-kilodalton (kDa) ?(6)?(6) dodecamers. Therefore the architecture of the MCC holoenzyme is expected to be highly similar to that of PCC. Here we report the crystal structures of the Pseudomonas aeruginosa MCC (PaMCC) holoenzyme, alone and in complex with coenzyme A. Surprisingly, the structures show that the architecture and overall shape of PaMCC are markedly different when compared to PCC. The ?-subunits show trimeric association in the PaMCC holoenzyme, whereas they have no contacts with each other in PCC. Moreover, the positions of the two domains in the ?-subunit of PaMCC are swapped relative to those in PCC. This structural information establishes a foundation for understanding the disease-causing mutations of MCC and provides new insights into the catalytic mechanism and evolution of biotin-dependent carboxylases. The large structural differences between MCC and PCC also have general implications for the relationship between sequence conservation and structural similarity.
Project description:Propionyl-coenzyme A carboxylase (PCC), a mitochondrial biotin-dependent enzyme, is essential for the catabolism of the amino acids Thr, Val, Ile and Met, cholesterol and fatty acids with an odd number of carbon atoms. Deficiencies in PCC activity in humans are linked to the disease propionic acidaemia, an autosomal recessive disorder that can be fatal in infants. The holoenzyme of PCC is an alpha(6)beta(6) dodecamer, with a molecular mass of 750 kDa. The alpha-subunit contains the biotin carboxylase (BC) and biotin carboxyl carrier protein (BCCP) domains, whereas the beta-subunit supplies the carboxyltransferase (CT) activity. Here we report the crystal structure at 3.2-A resolution of a bacterial PCC alpha(6)beta(6) holoenzyme as well as cryo-electron microscopy (cryo-EM) reconstruction at 15-A resolution demonstrating a similar structure for human PCC. The structure defines the overall architecture of PCC and reveals unexpectedly that the alpha-subunits are arranged as monomers in the holoenzyme, decorating a central beta(6) hexamer. A hitherto unrecognized domain in the alpha-subunit, formed by residues between the BC and BCCP domains, is crucial for interactions with the beta-subunit. We have named it the BT domain. The structure reveals for the first time the relative positions of the BC and CT active sites in the holoenzyme. They are separated by approximately 55 A, indicating that the entire BCCP domain must translocate during catalysis. The BCCP domain is located in the active site of the beta-subunit in the current structure, providing insight for its involvement in the CT reaction. The structural information establishes a molecular basis for understanding the large collection of disease-causing mutations in PCC and is relevant for the holoenzymes of other biotin-dependent carboxylases, including 3-methylcrotonyl-CoA carboxylase (MCC) and eukaryotic acetyl-CoA carboxylase (ACC).
Project description:<h4>Background</h4>Biotin-dependent carboxylases are a diverse family of carboxylating enzymes widespread in the three domains of life, and thus thought to be very ancient. This family includes enzymes that carboxylate acetyl-CoA, propionyl-CoA, methylcrotonyl-CoA, geranyl-CoA, acyl-CoA, pyruvate and urea. They share a common catalytic mechanism involving a biotin carboxylase domain, which fixes a CO? molecule on a biotin carboxyl carrier peptide, and a carboxyl transferase domain, which transfers the CO? moiety to the specific substrate of each enzyme. Despite this overall similarity, biotin-dependent carboxylases from the three domains of life carrying their reaction on different substrates adopt very diverse protein domain arrangements. This has made difficult the resolution of their evolutionary history up to now.<h4>Results</h4>Taking advantage of the availability of a large amount of genomic data, we have carried out phylogenomic analyses to get new insights on the ancient evolution of the biotin-dependent carboxylases. This allowed us to infer the set of enzymes present in the last common ancestor of each domain of life and in the last common ancestor of all living organisms (the cenancestor). Our results suggest that the last common archaeal ancestor had two biotin-dependent carboxylases, whereas the last common bacterial ancestor had three. One of these biotin-dependent carboxylases ancestral to Bacteria most likely belonged to a large family, the CoA-bearing-substrate carboxylases, that we define here according to protein domain composition and phylogenetic analysis. Eukaryotes most likely acquired their biotin-dependent carboxylases through the mitochondrial and plastid endosymbioses as well as from other unknown bacterial donors. Finally, phylogenetic analyses support previous suggestions about the existence of an ancient bifunctional biotin-protein ligase bound to a regulatory transcription factor.<h4>Conclusions</h4>The most parsimonious scenario for the early evolution of the biotin-dependent carboxylases, supported by the study of protein domain composition and phylogenomic analyses, entails that the cenancestor possessed two different carboxylases able to carry out the specific carboxylation of pyruvate and the non-specific carboxylation of several CoA-bearing substrates, respectively. These enzymes may have been able to participate in very diverse metabolic pathways in the cenancestor, such as in ancestral versions of fatty acid biosynthesis, anaplerosis, gluconeogenesis and the autotrophic fixation of CO?.
Project description:Genes for two subunits of acetyl-coenzyme A carboxylase, biotin carboxylase and biotin carboxyl carrier protein, have been cloned from Anabaena sp. strain PCC 7120. The two proteins are 181 and 447 amino acids long and show 40 and 57% identity to the corresponding Escherichia coli proteins, respectively. The sequence of the biotinylation site in Anabaena sp. strain PCC 7120 is MetLysLeu, not the MetLysMet found in other sequences of biotin-dependent carboxylases. The amino acid sequence of biotin carboxylase is also very similar (32 to 47% identity) to the sequence of the biotin carboxylase domain of other biotin-dependent carboxylases. Genes for these two subunits of acetyl-coenzyme A carboxylase are not linked in Anabaena sp. strain PCC 7120, contrary to the situation in E. coli, in which they are in one operon.
Project description:Human holocarboxylase synthetase (HCS) catalyzes linkage of the vitamin biotin to the biotin carboxyl carrier protein (BCCP) domain of five biotin-dependent carboxylases. In the two-step reaction, the activated intermediate, bio-5'-AMP, is first synthesized from biotin and ATP, followed by covalent linkage of the biotin moiety to a specific lysine residue of each carboxylase BCCP domain. Selectivity in HCS-catalyzed biotinylation to the carboxylases was investigated in single turnover stopped flow and quench flow measurements of biotin transfer to the minimal biotin acceptor BCCP fragments of the carboxylases. The results demonstrate that biotinylation of the BCCP fragments of the mitochondrial carboxylases propionyl-CoA carboxylase, pyruvate carboxylase, and methylcrotonoyl-CoA carboxylase is fast and limited by the bimolecular association rate of the enzyme with substrate. By contrast, biotinylation of the acetyl-CoA carboxylase 1 and 2 (ACC1 and ACC2) fragments, both of which are accessible to HCS in the cytoplasm, is slow and displays a hyperbolic dependence on substrate concentration. The correlation between HCS accessibility to biotin acceptor substrates and the kinetics of biotinylation suggests that mitochondrial carboxylase sequences evolved to produce fast association rates with HCS in order to ensure biotinylation prior to mitochondrial import. In addition, the results are consistent with a role for HCS specificity in dictating biotin distribution among carboxylases.
Project description:Holocarboxylase synthetase (HCS) catalyzes the binding of the vitamin biotin to carboxylases and histones. Carboxylases mediate essential steps in macronutrient metabolism. For example, propionyl-CoA carboxylase (PCC) catalyzes the carboxylation of propionyl-CoA in the metabolism of odd-chain fatty acids. HCS comprises four putative domains, i.e., the N-terminus, the biotin transfer/ATP-binding domain, a putative linker domain, and the C-terminus. Both N- and C-termini are essential for biotinylation of carboxylases by HCS, but the exact functions of these two domains in enzyme catalysis are unknown. Here we tested the hypothesis that N- and C-termini play roles in substrate recognition by HCS. Yeast-two-hybrid (Y2H) assays were used to study interactions between the four domains of human HCS with p67, a PCC-based polypeptide and HCS substrate. Both N- and C-termini interacted with p67 in Y2H assays, whereas the biotin transfer/ATP-binding and the linker domains did not interact with p67. The essentiality of N- and C-termini for interactions with carboxylases was confirmed in rescue experiments with mutant Saccharomyces cerevisiae, using constructs of truncated human HCS. Finally, a computational biology approach was used to model the 3D structure of human HCS and identify amino acid residues that interact with p67. In silico predictions were consistent with observations from Y2H assays and yeast rescue experiments, and suggested docking of p67 near Arg508 and Ser515 within the central domain of HCS.
Project description:3-Methylcrotonylglycinuria is an organic aciduria resulting from deficiency of 3-methylcrotonyl-CoA carboxylase (3-MCC), a biotin-dependent mitochondrial enzym carboxylating 3-methylcrotonyl-CoA to 3-methylglutaconyl-CoA during leucine catabolism. Its deficiency, due to mutations on MCCC1 and MCCC2 genes, leads to accumulation of 3-methylcrotonyl-CoA metabolites in blood and/or urine, primarily 3-hydroxyisovaleryl-carnitine (C5-OH) in plasma and 3-methylcrotonyl-glycine (3-MCG) and 3-hydroxyisovaleric acid (3-HIVA) in the urine. The phenotype of 3-MCC deficiency is highly variable, ranging from severe neurological abnormalities and death in infancy to asymptomatic adults. Here we report the biochemical and molecular characterization of an Italian asymptomatic girl, positive for the newborn screening test. Molecular analysis showed two mutations in the MCCC2 gene, an already described missense mutation, c.691A > T (p.I231F), and a novel splicing mutation, c.1150-1G > A. We characterized the expression profile of the splice mutation by functional studies.
Project description:We have cloned a DNA fragment from a genomic library of Myxococcus xanthus using an oligonucleotide probe representing conserved regions of biotin carboxylase subunits of acetyl coenzyme A (acetyl-CoA) carboxylases. The fragment contained two open reading frames (ORF1 and ORF2), designated the accB and accA genes, capable of encoding a 538-amino-acid protein of 58.1 kDa and a 573-amino-acid protein of 61.5 kDa, respectively. The protein (AccA) encoded by the accA gene was strikingly similar to biotin carboxylase subunits of acetyl-CoA and propionyl-CoA carboxylases and of pyruvate carboxylase. The putative motifs for ATP binding, CO(2) fixation, and biotin binding were found in AccA. The accB gene was located upstream of the accA gene, and they formed a two-gene operon. The protein (AccB) encoded by the accB gene showed high degrees of sequence similarity with carboxyltransferase subunits of acetyl-CoA and propionyl-CoA carboxylases and of methylmalonyl-CoA decarboxylase. Carboxybiotin-binding and acyl-CoA-binding domains, which are conserved in several carboxyltransferase subunits of acyl-CoA carboxylases, were found in AccB. An accA disruption mutant showed a reduced growth rate and reduced acetyl-CoA carboxylase activity compared with the wild-type strain. Western blot analysis indicated that the product of the accA gene was a biotinylated protein that was expressed during the exponential growth phase. Based on these results, we propose that this M. xanthus acetyl-CoA carboxylase consists of two subunits, which are encoded by the accB and accA genes, and occupies a position between prokaryotic and eukaryotic acetyl-CoA carboxylases in terms of evolution.
Project description:Biotin-dependent carboxylases are widely distributed in nature and have important functions in the metabolism of fatty acids, amino acids, carbohydrates, cholesterol and other compounds. Defective mutations in several of these enzymes have been linked to serious metabolic diseases in humans, and acetyl-CoA carboxylase is a target for drug discovery in the treatment of diabetes, cancer and other diseases. Here we report the identification and biochemical, structural and functional characterizations of a novel single-chain (120 kDa), multi-domain biotin-dependent carboxylase in bacteria. It has preference for long-chain acyl-CoA substrates, although it is also active towards short-chain and medium-chain acyl-CoAs, and we have named it long-chain acyl-CoA carboxylase. The holoenzyme is a homo-hexamer with molecular mass of 720 kDa. The 3.0 Å crystal structure of the long-chain acyl-CoA carboxylase holoenzyme from Mycobacterium avium subspecies paratuberculosis revealed an architecture that is strikingly different from those of related biotin-dependent carboxylases. In addition, the domains of each monomer have no direct contact with each other. They are instead extensively swapped in the holoenzyme, such that one cycle of catalysis involves the participation of four monomers. Functional studies in Pseudomonas aeruginosa suggest that the enzyme is involved in the utilization of selected carbon and nitrogen sources.
Project description:3-Methylcrotonylglycinuria is an inborn error of leucine catabolism and has a recessive pattern of inheritance that results from the deficiency of 3-methylcrotonyl-CoA carboxylase (MCC). The introduction of tandem mass spectrometry in newborn screening has revealed an unexpectedly high incidence of this disorder, which, in certain areas, appears to be the most frequent organic aciduria. MCC, an heteromeric enzyme consisting of alpha (biotin-containing) and beta subunits, is the only one of the four biotin-dependent carboxylases known in humans that has genes that have not yet been characterized, precluding molecular studies of this disease. Here we report the characterization, at the genomic level and at the cDNA level, of both the MCCA gene and the MCCB gene, encoding the MCC alpha and MCC beta subunits, respectively. The 19-exon MCCA gene maps to 3q25-27 and encodes a 725-residue protein with a biotin attachment site; the 17-exon MCCB gene maps to 5q12-q13 and encodes a 563-residue polypeptide. We show that disease-causing mutations can be classified into two complementation groups, denoted "CGA" and "CGB." We detected two MCCA missense mutations in CGA patients, one of which leads to absence of biotinylated MCC alpha. Two MCCB missense mutations and one splicing defect mutation leading to early MCC beta truncation were found in CGB patients. A fourth MCCB mutation also leading to early MCC beta truncation was found in two nonclassified patients. A fungal model carrying an mccA null allele has been constructed and was used to demonstrate, in vivo, the involvement of MCC in leucine catabolism. These results establish that 3-methylcrotonylglycinuria results from loss-of-function mutations in the genes encoding the alpha and beta subunits of MCC and complete the genetic characterization of the four human biotin-dependent carboxylases.