Donald M. Mock, MD, PhD and Nell I. Matthews, BA∗ Biotin contains two five-member rings made from a ureido group attached to a tetrahydrothiophene ring (Figure 26-1). A valeric acid side chain is attached to the tetrahydrothiophene ring. The pathway for biosynthesis of biotin was largely elaborated by M.A. Eisenberg and associates working with Escherichia coli (Eisenberg, 1973). Biotin is attached to the inactive apocarboxylase protein by a condensation reaction catalyzed by holocarboxylase synthetase (HCS) (see Figure 26-1). An amide bond is formed between the carboxyl group of the valeric acid side chain of biotin and the ε-amino group of a specific lysine residue in each of the apocarboxylases. These regions of each apocarboxylase contain sequences of amino acids (e.g., Met-Lys-Met at the attachment site) that are highly conserved for the individual carboxylases, and both the N- and C-terminus in HCS are important for recognition of the apocarboxylases (Hassan et al., 2009). In the carboxylase reaction, the carboxyl moiety is first attached to the biotin cofactor (of the holocarboxylase enzyme) at the ureido nitrogen opposite the side chain (Figure 26-2). Dehydration of HCO3− (bicarbonate) to CO2, forming carboxyphosphate, is driven by the hydrolysis of ATP to ADP (see Figure 26-2). The carboxyphosphate then carboxylates a specific nitrogen (N1) in the ureido containing ring of biotin to form N1 carboxybiotinyl-enzyme, and inorganic phosphate is released. These two steps allow HCO3−, which is present at a higher concentration in the cell fluid than is CO2, to be used for formation of the bound, chemically reactive form of CO2. The carboxyl group can then be transferred to a substrate yielding a carboxylated product. Because the valeric acid side chain of biotin is coupled to the side chain of lysine in each holocarboxylase, this CO2 is at the end of a long, flexible chain, allowing the biotinyl coenzyme to be carboxylated at one site and used as a CO2 donor at a second site. Methylcrotonyl-CoA carboxylase (MCC) catalyzes an essential step in the degradation of the branched-chain amino acid leucine (see Figure 26-4). MCC is composed of two nonidentical subunits: a biotinylated α subunit encoded by the gene MCCC1 and a nonbiotinylated β subunit encoded by the gene MCCC2. MCC is not regulated by small molecules or by dietary or hormonal factors. Deficient activity of MCC leads to metabolism of 3-methylcrotonyl-CoA to 3-hydroxyisovaleric acid, 3-hydroxyisovalerylcarnitine, and 3-methylcrotonylglycine by an alternate pathway (Stratton et al., 2010). Thus increased urinary excretion of these abnormal metabolites reflects deficient activity of MCC. The core histones are designated H2A, H2B, H3, and H4; each nucleosome contains one H3/H3/H4/H4 tetramer and two H2A/H2B dimers. Mammals also express a fifth class of histones: histone H1, which serves as a linker between nucleosomes. Each core histone has a globular domain and a flexible N-terminal tail. The N-terminal tails of core histones protrude from the nucleosomal surface, and covalent modifications of these tails play critical roles in gene regulation (Camporeale et al., 2004; Chew et al., 2006; Kobza et al., 2008). In 1995 Hymes and Wolf discovered that biotinidase can act as a biotinyl-transferase; biocytin serves as the source of biotin, and histones are specifically biotinylated (Hymes et al., 1995). Zempleni and co-workers reported that the abundance of biotinylated histones varies with the cell cycle and that biotinylated histones are increased approximately twofold in activated, dividing lymphocytes compared to quiescent lymphocytes (Stanley et al., 2002). These observations suggested that biotinylation of histones might play a role in regulating DNA transcription as an additional element in the “histone code.” Based on this initial work, it was believed that biotinylation of histones was catalyzed by biotinidase (Hymes et al., 1995). Indeed, approximately 25% of total cellular biotinidase activity is located in the nucleus, and histones are biotinylated enzymatically in a process that is catalyzed in vitro by biotinidase. However, subsequent studies provided evidence that HCS is substantially more important than biotinidase for biotinylation of histones in vivo (Chew et al., 2008; Gralla et al., 2008; Kobza et al., 2008). Fibroblasts from patients with inherited deficiency of HCS exhibit decreased biotinylation of histones. In contrast, biotin can be removed from histones by biotinidase, and debiotinylation of histones is decreased in samples from biotinidase-deficient patients. Current understanding is that HCS plays the predominant role in histone biotinylation and biotinidase plays the predominant role in histone debiotinylation. Although the mechanisms remain to be elucidated, biotin status clearly affects gene expression. Cell culture studies suggest that cell proliferation generates an increased demand for biotin, perhaps stemming from increased synthesis of biotin-dependent carboxylases. Evidence is emerging that this demand is met by an upregulation of biotin transporter expression that is mediated by biotinylation of lysine 12 in histone H4 (H4K12bio) (Gralla et al., 2008). Another function of H4K12bio appears to be chromosomal stability (Camporeale et al., 2007; Chew et al., 2008; Gralla et al., 2008; Wijeratne et al., 2010). Low levels of histone biotinylation have been reported in biotin-deficient cells and model organisms (Chew et al., 2008); reduced histone biotinylation has been linked to increased frequency of retrotransposition events, consistent with a role for histone biotinylation in chromosomal stability. Other roles for biotin in affecting gene expression have been observed. Pioneering studies by Dakshinamurti and co-workers reported a role for biotin in the regulation of the glucokinase gene (Dakshinamurti and Cheah-Tan, 1968). Solorzano-Vargas and colleagues (2002) reported that biotin deficiency reduces messenger RNA (mRNA) levels of HCS, ACC1, and PCC and postulated that a cyclic GMP–dependent signaling pathway is involved in the pathogenesis. Zempleni and co-workers have demonstrated involvement of nitric oxide in the cyclic GMP signaling pathway (Rodriguez-Melendez and Zempleni, 2009; Zempleni et al., 2009).
Biotin and Pantothenic Acid
Biotin Synthesis
Holocarboxylases and Holocarboxylase Synthetase
Biotin-Containing Carboxylases
Carboxylase Mechanism
Methylcrotonyl-Coa Carboxylase
Biotin and Gene Regulation
Biotinylation of Histones
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