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Supplement

Molecular Biology of Biotin Attachment to Proteins

Departments of Microbiology and Biochemistry, University of Illinois, Urbana, Illinois 61801

	ABSTRACT

TOP ABSTRACT INTRODUCTION BACTERIAL BIOTIN PROTEIN... THE CATALYTIC SITE REPRESSOR FUNCTION REGULATION OF BIOTIN... INTERACTION OF BIOTIN LIGASE... BIOTIN PROTEIN LIGASES OF... INTRACELLULAR LOCATION OF BPL MULTIPLE CARBOXYLASE DEFICIENCY REFERENCES

 
Enzymatic attachment of biotin to proteins requires the interaction of a distinct domain of the acceptor protein (the "biotin domain") with the enzyme, biotin protein ligase, that catalyzes this essential and rare post-translational modification. Both biotin domains and biotin protein ligases are very strongly conserved throughout biology. This review concerns the protein structures and mechanisms involved in the covalent attachment of biotin to proteins.

KEY WORDS: • biotin • domain • acceptor • ligase • post-translational

	INTRODUCTION

TOP ABSTRACT INTRODUCTION BACTERIAL BIOTIN PROTEIN... THE CATALYTIC SITE REPRESSOR FUNCTION REGULATION OF BIOTIN... INTERACTION OF BIOTIN LIGASE... BIOTIN PROTEIN LIGASES OF... INTRACELLULAR LOCATION OF BPL MULTIPLE CARBOXYLASE DEFICIENCY REFERENCES

 
Biotin (vitamin H), an essential coenzyme synthesized by plants and most procaryotes, is required by all organisms. In cells, biotin in its physiologically active form is covalently attached at the active site of a class of important metabolic enzymes, the biotin carboxylase and decarboxylases (Knowles 1989 , Samols et al. 1988 ). The biotin carboxylases are key enzymes in gluconeogenesis, lipogenesis, amino acid metabolism and energy transduction. These enzymes generally capture CO₂ from bicarbonate and catalyze transfer of this carboxylate to organic acids to form various cellular metabolites (Samols et al. 1988 ) by using the biotin cofactor as a mobile carboxyl carrier. Biotin protein ligase (BPL),⁴also known as holocarboxylase synthetase (EC 6.3.4.15), is the enzyme responsible for the covalent attachment of biotin to the cognate proteins. Biotin is attached post-translationally by BPL via an amide linkage to a specific lysine residue of newly synthesized carboxylases in a two-step reaction (Fig. 1 ).Although BPL is traditionally also referred to as a holocarboxylase synthetase, this name can be somewhat misleading because some biotinylated proteins are decarboxylases or transcarboxylases. BPL have also been named by the enzyme substrate used to assay BPL activity (as in holopyruvate carboxylase synthetase) in the belief that each biotin-dependent enzyme had a specific ligase. However, genetic studies in microorganisms and humans indicate that each organism has a single BPL-encoding gene. Moreover, this conclusion is consistent with the complete genome sequences available to date.

View larger version (13K): [in this window] [in a new window]   Figure 1. The biotin protein ligase (BPL) reaction.

	BACTERIAL BIOTIN PROTEIN LIGASES: STRUCTURE AND FUNCTION

TOP ABSTRACT INTRODUCTION BACTERIAL BIOTIN PROTEIN... THE CATALYTIC SITE REPRESSOR FUNCTION REGULATION OF BIOTIN... INTERACTION OF BIOTIN LIGASE... BIOTIN PROTEIN LIGASES OF... INTRACELLULAR LOCATION OF BPL MULTIPLE CARBOXYLASE DEFICIENCY REFERENCES

 
BPL from several organisms has been studied biochemically and genetically for some time. The first coding region to be isolated and sequenced was for the 35.5-kDa enzyme from E. coli (Barker and Campbell 1981b , Howard et al. 1985 ). More recently, sequences for the Bacillus subtilis homologue (Bower et al. 1995 ) and many other homologues have been annotated in the genomic sequences now becoming available (Fig. 2 ).The bifunctional nature of the BirA protein from E. coli was initially inferred from the localization of mutations within the birA gene. An extensive genetic analysis by Barker and Campbell (1981a and 1981b ) isolated a number of birA mutants that were either defective in repressor function or that increased the minimum requirement for biotin. Copurification of BPL activity with a protein that bound to bio operator DNA indicated that the ligase was a multifunctional protein that also acted as the transcriptional repressor controlling biotin biosynthesis (Barker and Campbell 1981b , Eisenberg et al. 1982 ). When the coding region was fully sequenced (Howard et al. 1985 ), the mutations affecting repressor function were shown to map to the N-terminal region of the protein, which contained a helix-turn-helix motif characteristic of DNA binding proteins (Buoncristiani et al. 1986 ); in contrast, those affecting the requirement for biotin and therefore the biotin ligase activity clustered in the center of the amino acid sequence. This sequence lies adjacent to a GRGRRG motif found at nucleotide binding sites (Buoncristiani et al. 1986 ). The other known bacterial genes also encode proteins of ~35 kDa which, from homology with the E. coli protein, have essentially the same domain structure. Several mutations that deregulate biotin biosynthesis have been localized within the DNA binding domain of the BirA protein from B. subtilis (Bower et al. 1995 ).

View larger version (87K): [in this window] [in a new window]   Figure 2. Comparison of the protein sequences of biotin protein ligase (BPL). Sequences for known BPL proteins of Escherichia coli, Bacillus subtilis, Arabidopsis thaliana, Saccharomyces cerevisiae and Homo sapiens and putative BPL from Paracoccus denitrificans, Schizosaccharomyces pombe, and Methanococcus janaschii were aligned over their entire length using Clustal W. Panel A: homologies within the BPL catalytic domain. Residues making contact with biotin in the crystal structure of E. coli BirA are marked with (Wilson et al. 1992) and the extent of the catalytic domain is indicated by the arrows. Highly conserved residues are shaded, highlighting both the GRGRR ATP-binding motif and the KWPND sequence also present in avidin (Tissot et al. 1997 ). Amino acid changes caused by mutations affecting BPL activity in E. coli BirA are indicated by (Buoncristiana et al. 1986) and those detected in patients with BPL deficiency are indicated by (Dupuis et al. 1996), with the substitutions indicated in bold. Panel B: homologies in the N-terminal region of eukaryotic BPL. Highly conserved residues are shaded. The positions of two altered amino acids detected in biotin-responsive BPL deficiency in humans are indicated by (Dupuis et al. 1996, Suzuki et al. 1994), with the substitutions indicated in bold.

	THE CATALYTIC SITE

TOP ABSTRACT INTRODUCTION BACTERIAL BIOTIN PROTEIN... THE CATALYTIC SITE REPRESSOR FUNCTION REGULATION OF BIOTIN... INTERACTION OF BIOTIN LIGASE... BIOTIN PROTEIN LIGASES OF... INTRACELLULAR LOCATION OF BPL MULTIPLE CARBOXYLASE DEFICIENCY REFERENCES

The mechanism of the biotin ligase reaction is straightforward (Fig. 1) and is similar to that of aminoacyl-tRNA synthetases. In the first half reaction, BPL catalyzes the attack of an oxygen atom of the biotin carboxylate on Pa of ATP to form biotinoyl-AMP (also called biotinoyl-adenylate) plus pyrophosphate. Biotinoyl-AMP remains bound in the active site and is quite stable when thus bound despite being a mixed anhydride. In the presence of the apoprotein (apo)-form of the biotin-accepting domain of a biotin-requiring enzyme, the nucleophilic -amino group of the lysine to be modified attacks the mixed anhydride carbon atom, thus forming the amide bond between biotin and the lysine side chain with AMP as the other product. Once the amide bond is formed, the biotin moiety remains attached throughout the lifetime of the protein molecule.

	REPRESSOR FUNCTION

TOP ABSTRACT INTRODUCTION BACTERIAL BIOTIN PROTEIN... THE CATALYTIC SITE REPRESSOR FUNCTION REGULATION OF BIOTIN... INTERACTION OF BIOTIN LIGASE... BIOTIN PROTEIN LIGASES OF... INTRACELLULAR LOCATION OF BPL MULTIPLE CARBOXYLASE DEFICIENCY REFERENCES

 
As predicted from the sequence data (Buoncristiani et al. 1986 ), the -helical N-terminal domain of the BirA protein has the helix-turn-helix structure of DNA-binding proteins, with a central DNA recognition helix (Wilson et al. 1992 ). Several birA mutants, defective in repressor function and having little effect on catalytic activity (Barker and Campbell 1981 ), map to the DNA recognition helix within the N-terminal domain, 40 Å removed from the biotin binding site (Wilson et al. 1992 ). Purified BirA was initially shown to bind to bio operator DNA by Barker and Campbell (1981b) . These workers and others (Prakash and Eisenberg 1979 ) also demonstrated that the biotin ligase enzymatic intermediate biotin-5'-AMP, not biotin, is the true corepressor. Thus, BirA is unique among DNA binding proteins in that it synthesizes its own corepressor.

BirA appears to undergo a number of conformational alterations related to repressor function. The 3-dimensional structure shows that the N-terminal DNA binding domain is connected to the rest of the molecule through a hinge, which would allow relocation of the domains in the course of the reaction (Beckett and Matthews 1997 , Wilson et al. 1992 ). A detailed thermodynamic study of the protein has shown that BirA exists in at least three distinct conformational states, depending on which of the ligands is bound (Xu et al. 1995 and 1996 , Xu and Beckett 1996 ). First, biotin binding causes a large structural change thought to facilitate ATP binding. After catalysis to form the corepressor, the protein undergoes further conformational change, presumably associated with formation of the DNA-binding conformation. Wilson et al. (1992) also concluded that large conformational changes are involved in substrate binding because attempts to make BirA-biotin or BirA-biotin-5'-AMP complexes resulted in destruction of the BirA crystals. In the operator-repressor complex, two repressor molecules bind to the biotin operator sequence, which is a 40-bp imperfect palindrome (Lin et al. 1991 , Lin and Shian 1993 , Streaker and Beckett 1998 ). However, BirA-biotin-5'-AMP in the absence of operator DNA is monomeric over the concentration range of DNA binding, and the binding of the second holorepressor molecule is cooperative (Streaker and Beckett 1998 ). Thus, DNA binding also involves a conformational change, allowing cooperative interaction between bound and unbound repressor molecules (Beckett and Matthews 1997 ). Another class of repressor mutations shown to lie >20 Å from the DNA recognition helix may affect conformational changes necessary for DNA binding or multimerization accompanying binding (Wilson et al. 1992 ).

	REGULATION OF BIOTIN BIOSYNTHESIS IN BACTERIA

TOP ABSTRACT INTRODUCTION BACTERIAL BIOTIN PROTEIN... THE CATALYTIC SITE REPRESSOR FUNCTION REGULATION OF BIOTIN... INTERACTION OF BIOTIN LIGASE... BIOTIN PROTEIN LIGASES OF... INTRACELLULAR LOCATION OF BPL MULTIPLE CARBOXYLASE DEFICIENCY REFERENCES

 
The biotin operon in E. coli contains five structural genes involved in biotin biosynthesis, with the operator located between two clusters (Fig. 3 )(Beckett and Matthews 1997 , Cronan 1989 ). Transcription of the operon is regulated, via BirA, by biotin concentration and apoprotein substrate supply, i.e., biotin biosynthesis is coupled to protein biotinylation (Cronan 1988 ). It is the biotin-5'-AMP level, rather than free biotin, that mediates the level of transcription (Cronan 1988 ), consistent with the biochemical data showing that biotin-5'-AMP is the corepressor (Abbott and Beckett 1993 , Barker and Campbell 1981b , Prakash and Eisenberg 1979 ). In fact, protein biotinylation and biotin uptake are closely coupled processes and intracellular biotin is predominantly protein bound (Barker and Campbell 1981a and 1981b , Cronan 1988). Because the intracellular concentration of ATP is in the millimolar range (Kornberg and Baker 1992 ), formation of corepressor and control of repressor function respond to the intracellular biotin concentration, which is considerably lower (10 nmol/L; Prakash and Eisenberg 1979). Thus, the ordered addition of biotin and ATP in the biotin ligase reaction appears to be a significant point of control in the regulation of biotin biosynthesis (Xu et al. 1995 ).

View larger version (22K): [in this window] [in a new window]   Figure 3. Regulation of transcription of the Escherichia coli biotin operon. The conditions shown are biotin-limited growth (panel A) and growth with biotin in excess (panel B). The asterisk (*) denotes the AMP moiety of biotinoyl-AMP. Figure modified from Cronan (1989) by inclusion of the results of Streaker and Beckett (1998). Abbreviations: The gene birA encodes BirA, the E. coli BPL. The gene accB encodes BCCP, the biotinylated subunit of E. coli acetyl-CoA carboxylase. The bioABCDF genes encode enzymes of biotin biosynthesis.

	INTERACTION OF BIOTIN LIGASE WITH THE PROTEIN SUBSTRATE

TOP ABSTRACT INTRODUCTION BACTERIAL BIOTIN PROTEIN... THE CATALYTIC SITE REPRESSOR FUNCTION REGULATION OF BIOTIN... INTERACTION OF BIOTIN LIGASE... BIOTIN PROTEIN LIGASES OF... INTRACELLULAR LOCATION OF BPL MULTIPLE CARBOXYLASE DEFICIENCY REFERENCES

 
The biotin carrier domain of the biotin-containing enzymes is commonly, but not invariably, located at the C-terminal end of the carboxylase, with the biotinyl-lysine ~35 residues from the C-terminus (Cronan 1990 , Samols et al 1988 ; Fig. 4 ).Although many of the biotin enzymes contain a single multifunctional polypeptide usually assembled into tetramers in the functional enzyme, several have the biotin carrier domain on a separate subunit (Samols et al. 1988 ). In transcarboxylase from Propionibacterium shermanii, which is an example of the latter class, biotinylation can occur before or after assembly of the subunits (Goss and Wood 1984 ). A high degree of similarity is apparent in the primary structure of biotin attachment domains of the many biotin carboxylases for which sequence data are now available. In particular, the specific biotinylated lysine residue occurs in a highly conserved AMKM tetrapeptide (Samols et al. 1988 ; Fig. 4 ). The results of a mutational analysis of the role of the Met residues flanking the biotin-lysine suggested that these residues are conserved because they are required for the carboxyl transfer reaction (Shenoy et al. 1992 ). In this study, primarily concerned with the carboxylase reaction, biotinylation was assessed qualitatively only. Quantitative data (Reche et al. 1998 ; S. Polyak,A. Chapman-Smith,J. E. Cronan,Jr. and J. C. Wallace,unpublished observations) now exist, indicating that the nature of the flanking methionines is also important for biotinylation. Thus the extreme conservation of the MKM motif is a significant part of both of the functional protein-protein interactions of the biotin domain.

View larger version (42K): [in this window] [in a new window]   Figure 4. Sequence alignment of the biotin domains of biotin carboxylases from diverse organisms. Residues forming ß-strands in the 3-dimensional structure of Escherichia coli biotin carboxyl carrier protein (BCCP) are underlined; hydrophobic core residues are indicated by (Athappily and Hendrickson 1995, Roberts 1996). The biotinylated lysine residue is marked . Shading indicates residues very highly conserved in all biotin domains for which sequence data are available. Positions at which amino acid substitution is known to reduce the efficiency of biotinylation are indicated by (Leon-Del-Rio et al. 1994, Murtif and Samols 1987). Alignment was done using Clustal W.

The 3-dimensional structure of the biotinylated form of the biotin domain of BCCP from E. coli has been determined recently by both nuclear magnetic resonance (NMR) (Roberts 1996 ) and X-ray crystallography (Athappily and Hendrickson 1995 ), giving essentially identical structures. The protein is a barrel structure consisting of two antiparallel ß-sheets each containing four strands, with the N- and C-termini close together at one end and the biotinyl-lysine exposed on a tight ß-turn on the opposite face of the molecule (Fig. 5 ).The sequence similarities around the biotin attachment site of biotin carboxylases from different sources are probably reflected in structural conservation because BPL will biotinylate acceptor proteins across a wide variety of species (Cronan 1990 , Leon-Del-Rio et al. 1995 , MacAllister and Coon 1966 , Tissot et al. 1996 ). Thus, it is anticipated that other biotin carrier proteins will have a highly homologous 3-dimensional structure. However, alignment of the sequences of biotin carrier proteins based on homology within the residues identified as being crucial to the formation of the hydrophobic core (Athappily and Hendrickson 1995 ) shows that the E. coli protein contains an additional sequence not generally found in other biotin domains (Fig. 4) ; it forms a protruding thumb in the 3-dimensional structure that interacts with the biotin moiety (Athappily and Hendrickson 1995 , Chapman-Smith et al. 1997 , Roberts 1996 ) (Fig. 5) . Although the function of this region is not known, it is clearly not essential for the biotinylation reaction because E. coli BPL will biotinylate apoproteins from which it is absent. Full-length BCCP has an additional N-terminal region of 70–80 residues, presumed to be the intersubunit interaction domain for assembly of the functional carboxylase, attached to the biotin carrier domain by a flexible Pro-Ala–rich linker region (Li and Cronan 1992 ).

View larger version (40K): [in this window] [in a new window]   Figure 5. The structure of the holodomain of biotin carboxyl carrier protein (BCCP) from E. coli. prepared using MOLSCRIPT (>Kraulis 1991 ) from the PDB coordinates of Athappily and Hendrickson (1995).

We have shown recently that a subtle global conformational change, detectable by sulfhydryl modification and limited proteolysis, accompanies biotinylation of the biotin domain from E. coli (Chapman-Smith et al. 1997 ). This change could be a consequence of the large structural changes that occur in the ligase molecule during reaction, and it may signal release of the biotinylated product. However, the solution structure for the apo- (unbiotinylated) protein determined by NMR analysis indicates that, in solution, the apoprotein has a structure very similar to that of the biotinylated domain with the same basic folding pattern, and only small localized differences are found between the two forms (Roberts 1996 , Yao et al. 1997 ). This implies that the altered properties upon biotinylation are due to differing dynamics of the two forms, but is unclear how this results in the extremely stable, virtually protease-resistant biotinylated form.

The results of several mutational analyses have begun to define residues in the biotin domain that are important for recognition of apoprotein by BPL. Not surprisingly, alteration of key hydrophobic core residues reduces the efficiency of biotinylation (Leon-Del-Rio and Gravel 1994 , Murtif and Samols 1987 ). Substitutions at several highly conserved glycine residues, which in the BCCP structure are involved in tight turns between ß-strands, also produce proteins that show poor biotinylation (Leon-Del-Rio and Gravel 1994 ; Chapman-Smith et al. 1999 ) These two classes of mutant proteins have destabilized structures, making them poor substrates for BPL. Using the biotin domain of human propionyl-CoA carboxylase, Leon-Del-Rio and Gravel (1994) showed that deleting the highly conserved PXXG motif found N-terminal to the biotin attachment site abolished biotinylation. Such a deletion within the core structural domain is likely to seriously disrupt folding. However, amino acid substitutions at these positions also severely reduced the efficiency of biotinylation (Leon-Del-Rio and Gravel 1994 ), implying that the sequence may be part of an important recognition epitope. Substituting Lys for Glu at two positions lying close to the biotinyl-lysine in the BCCP structure had little effect on the structure of the protein but dramatically reduced the efficiency of biotinylation (Chapman-Smith et al. 1999 ) Interestingly, the biotin binding pocket in BirA contains three basic residues (Wilson et al. 1992 ). Thus, charge conservation around the biotinylated lysine may be crucial for correct positioning of the two proteins. Further investigation of significant residues in both BPL and biotin acceptor domains is required to understand the nature of the protein-protein interactions involved in recognition and specificity.

	BIOTIN PROTEIN LIGASES OF EUKARYOTIC ORGANISMS

TOP ABSTRACT INTRODUCTION BACTERIAL BIOTIN PROTEIN... THE CATALYTIC SITE REPRESSOR FUNCTION REGULATION OF BIOTIN... INTERACTION OF BIOTIN LIGASE... BIOTIN PROTEIN LIGASES OF... INTRACELLULAR LOCATION OF BPL MULTIPLE CARBOXYLASE DEFICIENCY REFERENCES

 
In the last few years, the primary structure of three eukaryotic BPL has been determined. Two groups have reported the coding sequence for human BPL. Suzuki et al. (1994) used primers derived from the amino acid sequence of purified bovine BPL protein to identify cDNA clones, whereas Leon-Del-Rio et al. (1995) used functional complementation of an E. coli birA mutant. The open reading frame in the human cDNAs encodes a 726-residue protein (predicted M_r 80.8 kDa), with two possible initiating methionines identified at the 5' end of the coding region (Leon-Del-Rio et al. 1995 ). The gene for BPL from Saccharomyces cerevisiae and the cDNA from Arabidopsis thaliana were both isolated by complementation of an E. coli birA mutant. The yeast gene encodes a protein of 690 amino acids (Cronan and Wallace 1995 ), whereas the plant ligase is intermediate in size between the bacterial and other eukaryotic enzymes, with a predicted molecular mass of 41.1 kDa (Tissot et al. 1997 ). The protein sequence of the plant BPL gene carries an N-terminal organelle targeting sequence (chloroplast or mitochondrial) and hence may be of procaryotic origin, consistent with its small size.

All three BPL sequences show significant homology to the bacterial proteins in their C-terminal halves, allowing identification of these segments as the catalytic domains (Fig. 2) . In particular, residues in contact with biotin in the crystal structure of E. coli BirA (Wilson et al. 1992 ) are invariant in all of the proteins, and those associated with mutations in BirA that cause an increased K_m for biotin are highly conserved (Cronan and Wallace, 1995 , Leon-Del-Rio et al. 1995 , Tissot et al., 1997 ). Furthermore, the GRGRRG motif associated with ATP binding occurs in close proximity to the biotin binding site in all cases. In addition, sequences showing homology to the biotin binding protein, avidin, were identified in the C-terminal regions of both the human and plant proteins, supporting the identification of the catalytic site. The approaches used to isolate these coding sequences highlight the extreme conservation of both the ligase structure and the functional interaction between the enzyme and its protein substrate throughout evolution. Genome sequencing indicates that this conservation can be extended to the third kingdom, the archea. The genomes of Methanococcus jannaschii, M. thermoautotrophicum and Archaeoglobus fulgidus contain sequences that encode proteins very similar to the BPL and to biotinylated proteins found elsewhere in nature. In the case of M. thermoautotrophicum, a biotinylated protein has been shown to be a subunit of pyruvate carboxylase (Mukhopadhyay et al. 1998 ).

None of the eukaryotic proteins contain sequences that suggest DNA binding activity (Fig. 2) . This is consistent with biotin metabolism in the different organisms. Unlike bacteria, both yeast and humans require an exogenous source of biotin and therefore would not be expected to repress a nonfunctional pathway. Plants, on the other hand, do synthesize biotin; however, the intracellular free biotin concentration is ~2000-fold greater than in bacteria, suggesting the absence of a strong repression mechanism (Tissot et al. 1997 ). Both the human and yeast BPL proteins contain large additional N-terminal domains that share some sequence similarities (Cronan and Wallace 1995 ). Although there is as yet little indication of the function of this region of the eukaryotic enzyme, several intriguing mutations in the human BPL gene that affect ligase activity are located in this domain, away from the presumed catalytic site (Dupois et al. 1996 , Suzuki et al. 1994 ) as discussed below.

Given the sequence information now available and prior analyses of enzyme reactions (Chiba et al. 1994 , Goss and Wood 1984 , Suzuki et al. 1994 ), it now seems probable that the catalytically active form of eukaryotic BPL proteins is monomeric, like the bacterial enzymes. The rat liver enzyme is reported to be dimeric, with a monomer molecular weight of 50 kDa determined by SDS-PAGE (Xia et al. 1994 ). However, catalytically active enzyme obtained on gel filtration (~100 kDa) is similar in size to the monomeric molecular weight predicted for both yeast and human ligase from the sequence data (76.4 and 80.8 kDa, respectively). Furthermore, because the bovine ligase was sufficiently homologous to human ligase to allow isolation of human cDNA with primers designed from the bovine amino acid sequence, it seems likely that the vertebrate enzymes will have similar structures. In fact, Suzuki et al. (1994) noted that their bovine ligase preparation initially contained immunoreactive material of M_r 85 kDa and that the catalytically active 64-kDa protein isolated was most probably due to N-terminal proteolysis during the purification procedure.

	INTRACELLULAR LOCATION OF BPL

TOP ABSTRACT INTRODUCTION BACTERIAL BIOTIN PROTEIN... THE CATALYTIC SITE REPRESSOR FUNCTION REGULATION OF BIOTIN... INTERACTION OF BIOTIN LIGASE... BIOTIN PROTEIN LIGASES OF... INTRACELLULAR LOCATION OF BPL MULTIPLE CARBOXYLASE DEFICIENCY REFERENCES

 
In contrast to yeast, where the known biotin carboxylases are located in the cytosol (Lim et al. 1987 ), the situation in higher eukaryotes is more complex. In mammals, only acetyl-CoA carboxylase is cytosolic; pyruvate, propionyl-CoA and methylcrotonyl-CoA carboxylases are located in the mitochondria (Nyan 1988 , Robinson et al. 1983 ). In pea leaves, different forms of the biotin carboxylases are found in chloroplasts, mitochondria and the cytosol (Tissot et al. 1996 and 1997 ). Consistent with the subcellular location of the protein substrate, biotin ligase activity has been reported in both cytosolic and mitochondrial fractions isolated from mammalian cells (Chiba et al. 1994 , Chang and Cohen 1983 ). Taroni and Rosenberg (1991) showed that propionyl-CoA carboxylase in rat liver cells could be biotinylated either in the cytosol or after transport into the mitochondria, presenting unequivocal evidence for the presence of BPL in both cellular compartments. These observations have led to speculation that there may be different forms of BPL in the different cellular compartments. Hence, either BPL is encoded by two separate genes, or a single gene produces both the cytosolic and organelle forms. In the latter case, targeting would be directed by differential transcription or alternative translation initiation sites (Surguchov 1988 ).

The observation that cells from BPL-deficient patients have decreased levels of all four biotin carboxylases (Wolf 1995 ), together with chromosomal localization of the isolated human BPL coding sequence to a single site (Suzuki et al. 1994 ), constitutes strong evidence for the existence of a single gene in humans. The mechanism for targeting BPL to the mitochondria or cytosol has not yet been elucidated. The cDNA clones isolated by Suzuki et al. (1994) apparently encode only a cytosolic protein, suggesting that targeting involves differential transcription. Leon-Del-Rio et al. (1995) found alternative splice forms among their human BPL cDNA clones, generating two possible in-frame initiating Met codons. Because both of these were present in several isolates, the use of alternative translation initiation sites to determine subcellular localization is also possible. However, because the "leader" sequence does not correspond to a known mitochondrial import signal, direct determination of the fate of the translation products is required (Leon-Del-Rio et al. 1995 ).

In plants, the mechanism that determines localization is unclear. Pea leaves contain two isoforms of BPL, with apparently different isoelectric points (pI). The predominant form is found in the cytosol, and the minor form is found in mitochondria and chloroplasts (Tissot et al. 1997 ). Whether these isoforms are encoded by one or two genes is not known. The bpl cDNA clone isolated from the plant species A. thaliana encodes an N-terminal presequence similar to known organelle targeting sequences; a second in-frame methionine codon downstream from the putative targeting sequence may direct synthesis of a cytosolic form from the same sequence (Tissot et al. 1997 ). This interpretation is supported by the observed production of two proteins of 41 and 37 kDa after in vitro transcription and translation from the bpl clone. Further analysis of the bpl gene(s) and its transcription and translation products in both the mammalian and plant systems is required to determine which mechanisms are involved in targeting.

	MULTIPLE CARBOXYLASE DEFICIENCY

TOP ABSTRACT INTRODUCTION BACTERIAL BIOTIN PROTEIN... THE CATALYTIC SITE REPRESSOR FUNCTION REGULATION OF BIOTIN... INTERACTION OF BIOTIN LIGASE... BIOTIN PROTEIN LIGASES OF... INTRACELLULAR LOCATION OF BPL MULTIPLE CARBOXYLASE DEFICIENCY REFERENCES

 
Defects in BPL activity have been identified as the cause of the neonatal onset inherited metabolic disorder, Multiple Carboxylase Deficiency (MCD). The pattern of metabolite accumulation found in affected children indicates loss of the functions normally carried out by biotin carboxylases, and the resulting toxicity precludes normal development (Wolf 1995 ). In all known cases, the disorder is biotin responsive, and it is assumed that other classes of lesions in the bpl gene are lethal in utero. Analysis of BPL activity in cultured fibroblasts from affected individuals showed that the enzyme has a greatly increased K_m for biotin (between 3- and 70-fold), and there is a strong negative correlation between the magnitude of the K_m defect and the age of onset of the disease. In most cases investigated, the K_m for ATP is around normal or slightly elevated (Wolf 1995 ).

The recent determination of the sequence encoding BPL in humans has allowed identification of the primary lesion in the gene in individuals with MCD. Seven point mutations causing single amino acid substitutions and one single base deletion resulting in premature termination, present only in heterozygotes, have been reported. Four of the point mutations alter residues in the vicinity of the presumptive biotin binding site as identified by homology with the E. coli protein; these are consistent with the observed altered affinity for biotin (Dupuis et al. 1996 ). Another class of mutations also responsive to biotin cluster in an N-terminal sequence (residues 215–240) and thus appear to define a second region involved in biotin binding or protein stability (Dupuis et al. 1996 , Suzuki et al. 1994 ). It is interesting to note that a catalytically active form of bovine BPL may be truncated by ~130 residues at the N-terminus (Suzuki et al. 1994 ), thus possibly defining the N-terminal extent of the proposed second biotin interaction domain. The severity of the disease caused by impaired BPL function highlights the importance of biotinylation as a fundamental process within cells.

	FOOTNOTES

 
¹ To whom correspondence should be addressed.

¹ Presented at the symposium "Nutrition, Biochemistry and Molecular Biology of Biotin" as part of Experimental Biology 98, April 18–22, 1998, San Francisco, CA. The symposium was sponsored by the American Society for Nutritional Sciences and was supported in part by an educational grant from Roche Vitamins and Fine Chemicals. Published as a supplement to The Journal of Nutrition. Guest editor for the symposium publication was Donald Mock, University of Arkansas for Medical Sciences, Arkansas Children's Hospital, Little Rock, AR.

² Permanent address: Department of Biochemistry, The University of Adelaide, Adelaide, South Australia, 5005 Australia.

³ Abbreviations used: apo, apoprotein; BCCP, biotin carboxyl carrier protein; BPL, biotin protein ligase; MCD, multiple carboxylase deficiency; NMR, nuclear magnetic resonance.

	REFERENCES

TOP ABSTRACT INTRODUCTION BACTERIAL BIOTIN PROTEIN... THE CATALYTIC SITE REPRESSOR FUNCTION REGULATION OF BIOTIN... INTERACTION OF BIOTIN LIGASE... BIOTIN PROTEIN LIGASES OF... INTRACELLULAR LOCATION OF BPL MULTIPLE CARBOXYLASE DEFICIENCY REFERENCES

 

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