Structure of human holocarboxylase synthetase gene and mutation spectrum of holocarboxylase synthetase deficiency

Holocarboxylase synthetase (HLCS) is an enzyme that catalyzes the incorporation of biotin into apo-carboxylases, and its deficiency causes biotin-responsive multiple carboxylase deficiency. The reported sequences of cDNA for human HLCS from liver, lymphocyte, and KG-1 myeloid cell lines differ at their 5' regions. To elucidate variations of the human HLCS mRNA and longer 5' cDNA ends, we performed screening of the human liver cDNA library and rapid amplification of the cDNA ends (RACE). Our results suggest the existence of three types of HLCS mRNA that start at different exons. The first type starts at exon 1, and the second type starts at exon 3, and both are found in various human tissues. The third type, corresponding to the cDNA from the KG-1 cell, starts at exon 2 of the HLCS gene. Various splicing patterns from exons 3-6 were also observed. None of the variations of cDNA found created a new initiation codon. Mutation screening from exons 6-14, therefore, was sufficient to detect amino acid changes in HLCS in patients. Our direct sequencing strategy for screening mutations in the HLCS gene revealed mutations in five Japanese patients and seven non-Japanese patients. Our analyses involving 12 Japanese and 13 non-Japanese patients and studies by others indicate that (1) there is no panethnically prevalent mutation; (2) the Arg508Trp, Gly581Ser, and Val550Met mutations are found in both Japanese and non-Japanese populations; (3) the IVS10+5G-->A mutation is predominant and probably a founder mutation in European patients; (4) the 655-656insA, Leu237Pro, and 780delG mutations are unique in Japanese patients; (5) the spectrum of the mutations in the HLCS gene may vary substantially among different ethnic groups.     

Substrate recognition characteristics of human holocarboxylase synthetase for biotin ligation

Holocarboxylase synthetase (HCS) is an essential enzyme that catalyzes the incorporation of biotin into apo carboxylase and the biotinylation of the four biotin-dependent carboxylases in the human cell. Deficiency of HCS results in decreased activity of these carboxylases and affects various metabolic processes. Despite the importance of this enzyme, the recognition mechanism of the biotinoyl domain by human HCS (hHCS) has remained unclear. We have developed a method to express hHCS in the baculovirus system and used it to purify catalytically active, full-length hHCS. NMR experiments on the biotinoyl domains from acetyl-CoA carboxylase indicate that when hHCS is added, it recognizes the MKM motif in human and in Escherichia coli with a preference to the human biotinoyl domain. In addition, hHCS can biotinylate the biotinoyl domains from human and E. coli acetyl-CoA carboxylase at similar rates compared to the E. coli biotin protein ligase, BirA, which reacts very slowly with the human biotinoyl domain. We propose that the hHCS has greater substrate acceptability, while the BirA has higher substrate specificity. These results provide insights into substrate recognition by hHCS, which can be distinguished from BirA in this respect.     

Microbial biotin protein ligases aid in understanding holocarboxylase synthetase deficiency

The attachment of biotin onto the biotin-dependent enzymes is catalysed by biotin protein ligase (BPL), also known as holocarboxylase synthase HCS in mammals. Mammals contain five biotin-enzymes that participate in a number of important metabolic pathways such as fatty acid biogenesis, gluconeogenesis and amino acid catabolism. All mammalian biotin-enzymes are post-translationally biotinylated, and therefore activated, through the action of a single HCS. Substrate recognition by BPLs occurs through conserved structural cues that govern the specificity of biotinylation. Defects in biotin metabolism, including HCS, give rise to multiple carboxylase deficiency (MCD). Here we review the literature on this important enzyme. In particular, we focus on the new information that has been learned about BPL's from a number of recently published protein structures. Through molecular modelling studies insights into the structural basis of HCS deficiency in MCD are discussed.     

Regulation and intracellular localization of the biotin holocarboxylase synthetase of 3T3-L1 cells

A quantitative assay has been developed to measure holocarboxylase synthetase activity in cellular extracts. This assay was based on measuring the incorporation of [3H]biotin of high specific activity (4.3 Ci/mmol) into purified rat liver apopyruvate carboxylase. With this assay, holocarboxylase synthetase in 3T3-L1 mouse fibroblasts has been monitored. During the differentiation of this cell from a fibroblast to an adipocyte, holocarboxylase synthetase activity was found to increase threefold, while pyruvate carboxylase activity rose 20-fold. The results suggest a possible relationship between the activity of the holocarboxylase synthetase and the level of the biotin-dependent carboxylases within the mammalian cell. Utilizing digitonin fractionation. the intracellular distribution of this enzyme has also been examined. In the 3T3-L1 cell, the large majority (approximately 70%) of the total holocarboxylase synthetase activity was found in the cytosolic compartment.     

Heterogeneity of holocarboxylase synthetase in patients with biotin-responsive multiple carboxylase deficiency.

Holocarboxylase synthetase activity has been determined in fibroblasts of seven patients with the neonatal form of biotin-responsive multiple carboxylase deficiency. The normal Km for biotin was 15 +/- 3 nmol/l, while in the patients the values ranged from 48 to 1,062 nmol/l. The mean maximum velocity was 27% of normal. Differences among the values obtained for the Km for biotin and the heat stability of holocarboxylase synthetase suggested that the patients studied represented at least four distinct variants at the holocarboxylase synthetase locus.     

The N-terminal domain of human holocarboxylase synthetase facilitates biotinylation via direct interaction with the substrate protein

Human holocarboxylase synthetase shows a high degree of sequence homology in the catalytic domain with bacterial biotin ligases such as Escherichia coli BirA, but differs in the length and sequence of the N-terminus. Despite several studies having been undertaken on the N-terminal region of hHCS, the role of this region remains unclear. We determined the structure of the N-terminal domain of hHCS by limited proteolysis and showed that this domain has a crucial effect on the enzymatic activity. The domain interacts not only with biotin acceptor protein, but also with the catalytic domain of hHCS, as shown by nuclear magnetic resonance (NMR) experiments. We propose that the N-terminal domain of hHCS recognizes the charged region of biotin acceptor protein, distinctly from the recognition by the catalytic domain.

Structured summary

MINT-7543113: hHCS (uniprotkb:P50747) and hHCS (uniprotkb:P50747) bind (MI:0407) by nuclear magnetic resonance (MI:0077)MINT-7543096, MINT-7543129: ACC75 (uniprotkb:O00763) and hHCS (uniprotkb:P50747) bind (MI:0407) by nuclear magnetic resonance (MI:0077)MINT-7543053: hHCS (uniprotkb:P50747) enzymaticly reacts (MI:0414) ACC75 (uniprotkb:O00763) by nuclear magnetic resonance (MI:0077)MINT-7543070: hHCS (uniprotkb:P50747) enzymaticly reacts (MI:0414) ACC75 (uniprotkb:O00763) by enzymatic study (MI:0415)     

Identification of holocarboxylase synthetase chromatin binding sites in human mammary cell lines using the DNA adenine methyltransferase identification technology

Holocarboxylase synthetase (HCS) is a chromatin protein that is essential for mediating the covalent binding of biotin to histones. Biotinylation of histones plays crucial roles in the repression of genes and repeats in the human genome. We tested the feasibility of DNA adenine methyltransferase identification (DamID) technology to map HCS binding sites in human mammary cell lines. Full-length HCS was fused to DNA adenine methyltransferase (Dam) for subsequent transfection into breast cancer (MCF-7) and normal breast (MCF-10A) cells. HCS docking sites in chromatin were identified by using the unique adenine methylation sites established by Dam in the fusion construct; docking sites were unambiguously identified using methylation-sensitive digestion, cloning, and sequencing. In total, 15 novel HCS binding sites were identified in the two cell lines, and the following 4 of the 15 overlapped between MCF-7 and MCF-10A cells: inositol polyphosphate-5-phosphatase A, corticotropin hormone precursor, ribosome biogenesis regulatory protein, and leptin precursor. We conclude that DamID is a useful technology to map HCS binding sites in human chromatin and propose that the entire set of HCS binding sites could be mapped by combining DamID with microarray technology.     

Molecular characterization of a second copy of holocarboxylase synthetase gene (hcs2) in Arabidopsis thaliana.

Holocarboxylase synthetase (HCS), catalyzing the covalent attachment of biotin, is ubiquitously represented in living organisms. Indeed, the biotinylation is a post-translational modification that allows the transformation of inactive biotin-dependent carboxylases, which are committed in fundamental metabolisms such as fatty acid synthesis, into their active holo form. Among other living organisms, plants present a peculiarly complex situation. In pea, HCS activity has been detected in three subcellular compartments and the systematic sequencing of the Arabidopsis genome revealed the occurrence of two hcs genes (hcs1 and hcs2). Hcs1 gene product had been previously characterized at molecular and biochemical levels. Here, by PCR amplification, we cloned an hcs2 cDNA from Arabidopsis thaliana (Ws ecotype) mRNA. We observed the occurrence of multiple cDNA forms which resulted from the alternative splicing of hcs2 mRNA. Furthermore, we evidenced a nucleotide polymorphism at the hcs2 gene within the Ws ecotype, which affected splicing of hcs2 mRNA. This contrasted sharply with the situation at hcs1 locus. However, this polymorphism had no apparent effect on total HCS activity in planta. Finally, hcs2 mRNAs were found 4-fold less abundant than hcs1 mRNA and the most abundant hcs2 mRNA spliced variant should code for a truncated protein. We discuss the possible role of such a multiplicity of putative HCS proteins in plants and discuss the involvement of each of hcs genes in the correct realization of biotinylation.     

The polypeptide Syn67 interacts physically with human holocarboxylase synthetase, but is not a target for biotinylation

Holocarboxylase synthetase (HCS) catalyzes the binding of biotin to lysines in carboxylases and histones in two steps. First, HCS catalyzes the synthesis of biotinyl-5′-AMP; second, the biotinyl moiety is ligated to lysine residues. It has been proposed that step two is fairly promiscuous, and that protein biotinylation may occur in the absence of HCS as long as sufficient exogenous biotinyl-5′-AMP is provided. Here, we identified a novel polypeptide (Syn67) with a basic patch of lysines and arginines. Yeast-two-hybrid assays and limited proteolysis assays revealed that both N- and C-termini of HCS interact with Syn67. A potential target lysine in Syn67 was biotinylated by HCS only after arginine-to-glycine substitutions in Syn67 produced a histone-like peptide. We identified a Syn67 docking site near the active pocket of HCS by in silico modeling and site-directed mutagenesis. Biotinylation of proteins by HCS is more specific than previously assumed.     

The role of holocarboxylase synthetase in genome stability is mediated partly by epigenomic synergies between methylation and biotinylation events

Holocarboxylase synthetase (HLCS) catalyzes the covalent binding of biotin to histones. Biotinylated histones are gene repression marks and are particularly enriched in long terminal repeats, telomeres and other repeat regions. The effects of HLCS in gene regulation are mediated by its physical interactions with chromatin proteins such as histone H3, DNMT1, MeCP2 and EHMT-1. It appears that histone biotinylation depends on prior methylation of cytosines. De-repression of long terminal repeats in biotin- or HLCS-deficient cell cultures and organisms is associated with genome instability.Key words: biotin, DNMT1, EHMT-1, genome stability, histone, holocarboxylase synthetase, MeCP2, methylation     

The role of holocarboxylase synthetase in genome stability is mediated partly by epigenomic synergies between methylation and biotinylation events

Holocarboxylase synthetase (HLCS) catalyzes the covalent binding of biotin to histones. Biotinylated histones are gene repression marks and are particularly enriched in long terminal repeats, telomeres, and other repeat regions. The effects of HLCS in gene regulation are mediated by its physical interactions with chromatin proteins such as histone H3, DNMT1, MeCP2, and EHMT-1. It appears that histone biotinylation depends on prior methylation of cytosines. De-repression of long terminal repeats in biotin- or HLCS-deficient cell cultures and organisms is associated with genome instability.     

Evidence for a defect of holocarboxylase synthetase activity in cultured lymphoblasts from a patient with biotin-responsive multiple carboxylase deficiency.

We report here the expression of biotin-responsive multiple carboxylase deficiency in cultured lymphoblasts of a patient whose fibroblasts belong to the bio genetic complementation group. Cultured lymphoblasts from the patient lost propionyl-CoA carboxylase (PCC) and beta-methylcrotonyl-CoA carboxylase (MCC) activities at a faster rate than normal cells when grown in biotin-deficient medium. Recovery of normal PCC and MCC activities, which was independent of protein synthesis, required a 2,500-fold higher biotin concentration than that required by normal lymphoblasts. Holocarboxylase synthetase activity was detected in cell-free extracts through the biotinylation of endogenous apo-PCC in the presence of ATP to form active holo-PCC. While the apo-PCC in extracts of normal biotin-starved lymphoblasts could be activated to 28% of maximal activity, extracts of patient lymphoblasts did not exhibit any ATP and biotin-dependent increase in PCC activity. A normal cell extract, cleared of apocarboxylases by immunoprecipitation, stimulated the PCC activity of a patient cell extract 20-fold. These results indicate that the apoenzyme in bio cells is normal and that the defect lies in the holocarboxylase synthetase.     

Dual targeting of Arabidopsis holocarboxylase synthetase1: a small upstream open reading frame regulates translation initiation and protein targeting

Protein biotinylation is an original and very specific posttranslational modification, compartmented in plants, between mitochondria, plastids, and the cytosol. This reaction modifies and activates few carboxylases committed in key metabolisms and is catalyzed by holocarboxylase synthetase (HCS). The molecular bases of this complex compartmentalization and the relative function of each of the HCS genes, HCS1 and HCS2, identified in Arabidopsis (Arabidopsis thaliana) are mainly unknown. Here, we showed by reverse genetics that the HCS1 gene is essential for plant viability, whereas disruption of the HCS2 gene in Arabidopsis does not lead to any obvious phenotype when plants are grown under standard conditions. These findings strongly suggest that HCS1 is the only protein responsible for HCS activity in Arabidopsis cells, including the cytosolic, mitochondrial, and plastidial compartments. A closer study of HCS1 gene expression enabled us to propose an original mechanism to account for this multiplicity of localizations. Located in the HCS1 messenger RNA 5'-untranslated region, an upstream open reading frame regulates the translation initiation of HCS1 and the subsequent targeting of HCS1 protein. Moreover, an exquisitely precise alternative splicing of HCS1 messenger RNA can regulate the presence and absence of this upstream open reading frame. The existence of these complex and interdependent mechanisms creates a rich molecular platform where different parameters and factors could control HCS targeting and hence biotin metabolism.     

Expression in Escherichia coli of N- and C-terminally deleted human holocarboxylase synthetase. Influence of the N-terminus on biotinylation and identification of a minimum functional protein

Biotin functions as a covalently bound cofactor of biotindependent carboxylases. Biotin attachment is catalyzed by biotin protein ligases, called holocarboxylase synthetase in mammals and BirA in prokaryotes. These enzymes show a high degree of sequence similarity in their biotinylation domains but differ markedly in the length and sequence of their N terminus. BirA is also the repressor of the biotin operon, and its DNA attachment site is located in its N terminus. The function of the eukaryotic N terminus is unknown. Holocarboxylase synthetase with N- and C-terminal deletions were evaluated for the ability to catalyze biotinylation after expression in Escherichia coli using bacterial and human acceptor substrates. We showed that the minimum functional protein is comprised of the last 349 of the 726-residue protein, which includes the biotinylation domain. Significantly, enzyme containing intermediate length, N-terminal deletions interfered with biotin transfer and interaction with different peptide acceptor substrates. We propose that the N terminus of holocarboxylase synthetase contributes to biotinylation through N- and C-terminal interactions and may affect acceptor substrate recognition. Our findings provide a rationale for the biotin responsiveness of patients with point mutations in the N-terminal sequence of holocarboxylase synthetase. Such mutant enzyme may respond to biotin-mediated stabilization of the substrate-bound complex.