Biotin, which serves as a carboxyl group carrier in reactions catalyzed by biotin-dependent carboxylases, is essential for life in most organisms. To function in carboxylate transfer, the vitamin must be post-translationally linked to a specific lysine residue on the biotin carboxyl carrier (BCC) of a carboxylase in a reaction catalyzed by biotin protein ligases. Although biotin addition is highly selective for any single carboxylase substrate, observations of interspecies biotinylation suggested little discrimination among the BCCs derived from the carboxylases of a broad range of organisms. Application of single turnover kinetic techniques to measurements of post-translational biotin addition reveals previously unappreciated selectivity that may be of physiological significance.

Biotin: a brief overview and history

Biotin, vitamin B7 or vitamin H, is an essential nutrient that is required by most organisms for viability [1]. It functions as a coenzyme in carboxylation [2] reactions that contribute to important mammalian metabolic processes, including fatty acid biosynthesis and breakdown, amino acid catabolism and gluconeogenesis. The mammalian biotin-dependent carboxylases include acetyl-CoA carboxylase (ACC), propionyl-CoA carboxylase (PCC), pyruvate carboxylase (PC) and methylcrotonyl-CoA carboxylase (MCC). Loss of function of any of these enzymes has serious consequences for human health [35].

Biotin was first identified in studies that revealed the toxic effects of feeding raw egg whites, which contain the biotin-binding protein avidin, to rats. These effects, which included skin lesions, alopecia and loss of muscle coordination, could be reversed by protective factor X, a heat stable factor from yeast or liver [6]. In 1927, Boas [6] showed that animals required factor X, which was also referred to as vitamin H. György et al. [7] determined that factor X, vitamin H and biotin were the same compound. The structure of biotin was determined in 1942 (Figure 1, [8]).

The structure of biotin.

Figure 1.
The structure of biotin.

Chemical structure (top) (ChemDraw) and three-dimensional model (bottom) of biotin (Pymol (74)).

Figure 1.
The structure of biotin.

Chemical structure (top) (ChemDraw) and three-dimensional model (bottom) of biotin (Pymol (74)).

Biotin-dependent carboxylases

Biotin-dependent carboxylases are complex machines that contain a minimum of three activities, including a biotin carboxyl carrier (BCC or BCCP), a biotin carboxylase (BC) and a carboxyl transferase (CT) (Figure 2A, [9]). The biotin coenzyme is linked to a specific lysine residue on the BCC moiety of the enzyme. In some biotin-dependent carboxylases, all three activities are encoded in a single polypeptide chain. In others, typically from prokaryotes, the activities can each be encoded on separate subunits. Alternatively, the BC and BCC activities are found on one subunit and the CT on a second. In the first half-reaction catalyzed by any carboxylase, which occurs at the BC active site, a carboxyl group from a donor molecule is covalently linked to the ureido nitrogen of the biotin coenzyme on BCC in an ATP-dependent reaction (Figure 2A, [10]). In the second half reaction, which occurs at the CT site, the carboxyl group is transferred to the acceptor molecule.

Structure and function of biotin-dependent carboxylases.

Figure 2.
Structure and function of biotin-dependent carboxylases.

(A) Biotin-dependent carboxylases catalyze the two-step transfer of carboxylate groups from a donor to an acceptor. In the first step, which takes place at the BC site, the carboxylate is linked to the N1′ of biotin in an ATP-dependent reaction. The second step, which takes place at the CT site, involves transfer of the carboxylate from biotin to an acceptor. (B) Three-dimensional structure of the S. cerevisiae Acetyl-CoA carboxylase homodimer. One monomer, on the backside of the dimer, is shown in grey. The dark teal segment connects the BC and CT domains. Domains ACC1-5, which are shown in pink, are thought to be important for regulating the enzyme activity. The model was created in Pymol [74] with pdb file 5CSL.

Figure 2.
Structure and function of biotin-dependent carboxylases.

(A) Biotin-dependent carboxylases catalyze the two-step transfer of carboxylate groups from a donor to an acceptor. In the first step, which takes place at the BC site, the carboxylate is linked to the N1′ of biotin in an ATP-dependent reaction. The second step, which takes place at the CT site, involves transfer of the carboxylate from biotin to an acceptor. (B) Three-dimensional structure of the S. cerevisiae Acetyl-CoA carboxylase homodimer. One monomer, on the backside of the dimer, is shown in grey. The dark teal segment connects the BC and CT domains. Domains ACC1-5, which are shown in pink, are thought to be important for regulating the enzyme activity. The model was created in Pymol [74] with pdb file 5CSL.

The Saccharomyces cerevisiae (S. cerevisiae) ACC provides one example of the complex architecture of the biotin-dependent carboxylases and its relationship to function [11]. This enzyme catalyzes the conversion of acetyl-CoA to malonyl-CoA, the first committed step of fatty acid biosynthesis. In the S. cerevisiae enzyme the BCC, BC and CT are encoded in a single polypeptide chain which self-associates to form a homodimer (Figure 2B). In the catalytic cycle, the biotin-bound BCC first engages with the BC active site to accept carboxylate from bicarbonate. Prior to the second step, in which the carboxyl group is transferred to acetyl-CoA to form malonyl-CoA, the BCC domain must translocate 80 Å to the CT domain. It is thought that the additional domains (AC1-5), shown in pink in the structural model, contribute to this translocation event. While other biotin-dependent carboxylases share domain structure similar to that of Sc ACC, the three-dimensional architectures are highly variable [12].

Post-translational biotin addition to carboxylases

In its coenzyme function in biotin-dependent carboxylase reactions biotin is covalently linked to the BCC domain via an amide linkage that is formed between the carboxyl group of the valerate chain of biotin and the ε-amino group of a single lysine residue on the BCC domain [13]. This two-step post-translational biotinylation reaction is catalyzed by biotin protein ligases (BPL, Figure 3A). In the first step, the biotin is activated by adenylation using ATP as a substrate [13]. The resulting enzyme-biotinyl-5′-AMP complex then associates with BCCP to catalyze amide bond formation and AMP release.

Two-step post-translational biotin addition catalyzed by BPLs.

Figure 3.
Two-step post-translational biotin addition catalyzed by BPLs.

(A) Step 1: Biotin is adenylated to yield the biotinoyl-5′-adenylated intermediate (bio-5′-AMP). Step 2: The enzyme:intermediate complex interacts with the BCC to catalyze formation of an amide linkage between biotin and the ε-amino group of a specific lysine. (B) Three classes of BPLs: Class 1(4OP0) and 2 (2EWN) BPLs are found in archaea and eubacteria and the Class 3 enzymes are present in eukaryotes. In the Class 3 enzymes, the N-terminal domain, shown as a grey oval, varies in size. The models were created using PyMol [74].

Figure 3.
Two-step post-translational biotin addition catalyzed by BPLs.

(A) Step 1: Biotin is adenylated to yield the biotinoyl-5′-adenylated intermediate (bio-5′-AMP). Step 2: The enzyme:intermediate complex interacts with the BCC to catalyze formation of an amide linkage between biotin and the ε-amino group of a specific lysine. (B) Three classes of BPLs: Class 1(4OP0) and 2 (2EWN) BPLs are found in archaea and eubacteria and the Class 3 enzymes are present in eukaryotes. In the Class 3 enzymes, the N-terminal domain, shown as a grey oval, varies in size. The models were created using PyMol [74].

Although BPLs from all three domains of life have been biochemically characterized [1417], structures of only archael and eubacterial enzymes are known [1820]. As indicated by the domain architectures of these enzymes, they share an ∼25 kDa segment that forms the basic catalytic unit (Figure 3B). The eukaryotic enzymes, based on sequence homology, possess this same structural unit. A subset of the enzymes from the archael and eubacterial families contain an N-terminal DNA-binding domain, which enables regulation of biotin biosynthesis and/or transport by transcription repression at the operons that encode these functions [1]. In addition to the N-terminal DNA-binding domain, some bacterial BPLs, such as those found in Neisseria species, possess enzyme encoding C-terminal domains. The N-terminal domains of eukaryotic BPLs are considerably larger than those of Class 2 enzymes [2123]. Although the sequences of these segments are suggestive of functions, little biochemical evidence supporting these functions exists. Mutations in the coding region for the N-terminal domain of the human enzyme are associated with defects in biotin transfer to carboxylase substrates [21]. Two of these mutations disrupt the protein:protein interaction that forms between the enzyme:intermediate and the BCC substrate in biotin transfer [24]

Some of the sequence and structural requirements on BCC for post-translational biotin addition have been defined. Results of early studies indicated that the activities of the intact carboxylases and the isolated BCC domains are equivalent in BPL-catalyzed biotin transfer [25]. Studies of the BCCP subunit of Escherichia coli (E. coli) Acetyl-CoA carboxylase indicate that an 84-residue segment provides the minimal substrate for EcBirA-catalyzed biotinylation [26]. In the Propionibacterium shermanii transcarboxylase 1.3S subunit 30–40 residues N- and C-terminal to the target lysine were required for activity in biotin addition [27]. Structural studies revealed that this segment folds into a topology that is described as a ‘capped β-sandwich' in which two 3-stranded antiparallel sheets form the sandwich sides and two single strands cap the sandwich (Figure 4, [28]). Consistent with predictions based on sequence alignment, subsequent studies revealed similar structures for BCC domains of carboxylases from bacteria to mammals [2932]. In addition to the overall structural conservation, the sequence of the BCC domains in the vicinity of the target lysine is highly conserved, with the lysine residue embedded between two methionines. However, amino acid substitutions of these methionines did not affect biotin transfer to the 1.3S subunit of P. shermanii transcarboxylase in vivo [33]. Rather, these residues appeared to function in carboxylate transfer to the biotin moiety in the first half reaction of the carboxylase. Studies of the biotinoyl domain of E. coli ACC indicate that substitution of these methionines does affect the E. coli BPL-catalyzed biotin addition [34].

Sequences and structures of BCC domains are conserved.

Figure 4.
Sequences and structures of BCC domains are conserved.

(A) Sequences of the BCC segments of Top: Human Acetyl-CoA carboxylase 2, Middle: E. coli Acetyl-CoA carboxylase, Bottom: P. shermanii Transcarboxylase. The red star indicates the target lysine. (B) Alignment of three-dimensional structures of BCC domains of Red: human ACC2 (pdb ID 2KCC), Purple: E. coli ACC (pdb ID 1BDO) and Yellow: P. shermanii TC (pdb ID 1DCC). The E. coli model includes the biotinylated lysine and is characterized by an ‘extra' segment designated a protruding thumb. The models were created and aligned in PyMol [74].

Figure 4.
Sequences and structures of BCC domains are conserved.

(A) Sequences of the BCC segments of Top: Human Acetyl-CoA carboxylase 2, Middle: E. coli Acetyl-CoA carboxylase, Bottom: P. shermanii Transcarboxylase. The red star indicates the target lysine. (B) Alignment of three-dimensional structures of BCC domains of Red: human ACC2 (pdb ID 2KCC), Purple: E. coli ACC (pdb ID 1BDO) and Yellow: P. shermanii TC (pdb ID 1DCC). The E. coli model includes the biotinylated lysine and is characterized by an ‘extra' segment designated a protruding thumb. The models were created and aligned in PyMol [74].

A high-resolution structure of only the BPL:BCC complex from P. horikoshii has been determined (Figure 5, [31]). In this structure, the interface between the enzyme and substrate protein is formed through hydrogen bonding between two parallel β-strands. Additionally, a limited number of inter-side-chain and side-chain–backbone interactions, primarily involving charged residues, are formed in the complex. The structure supports the importance of specific residues that are known to function in E. coli BPL-catalyzed reaction. For example, alanine substitutions of the K, N or D in the KWPND sequence that is universally conserved in BPLs, have large effects on bio-5′-AMP synthesis and/or biotin transfer from the intermediate to BCCP [35]. This sequence forms a loop segment that is located in the enzyme active site. In addition, substitution of K194 in the E. coli enzyme results in a 7-fold increase in the Km for apoBCCP in the overall two-step biotin transfer reaction [35]. The homologous residue in the P. horikoshii complex forms a charged hydrogen bond with a backbone carbonyl of the BCCP domain. The structure is also consistent with the drastic effect of substituting EcBCC residue E119 with K, equivalent to E112 in the PhBCC, on biotin addition [36]. In the structure, the glutamate side chain forms an electrostatic interaction with the target lysine that may be important for the reaction.

Figure 5.

Three-dimensional structure of the P. horikoshii BPL:BCCP complex with the BCCP shown in blue and the BPL in the color scheme used in Figure 3 . The yellow segment highlights the loop comprised of residues KWPND, which are universally conserved in BPLs. The target lysine side chain (blue), adenosine and biotin (black) are shown in stick representation. The figure was created in PyMol [74] using pdb file 2EJG as input.

Figure 5.

Three-dimensional structure of the P. horikoshii BPL:BCCP complex with the BCCP shown in blue and the BPL in the color scheme used in Figure 3 . The yellow segment highlights the loop comprised of residues KWPND, which are universally conserved in BPLs. The target lysine side chain (blue), adenosine and biotin (black) are shown in stick representation. The figure was created in PyMol [74] using pdb file 2EJG as input.

Specificity in BPL-catalyzed biotin addition

In vivo post-translational biotin addition is a highly specific reaction, with only a limited number of proteins targeted in any one organism. For example, in E. coli only the BCCP subunit of ACC is biotinylated [37]. In mammalian cells biotin is linked to only the five carboxylases [38,39]. In spite of this physiological specificity, results of several studies suggest a lack of specificity.

The interspecies biotinylation observed for both eukaryotic and prokaryotic BPLs suggests low target specificity. For example, the enzymes from diverse sources, including rat liver, chicken liver, yeast and bacteria catalyze biotin addition to apocarboxylases from other species [40,41]. Cronan demonstrated that the E. coli BPL, BirA, can biotinylate the BCC moieties from several bacteria and even from tomato [27]. Furthermore, cloning of the BPLs from diverse organisms, including heterologous bacteria, humans and fungi was accomplished by complementation of a birAts E. coli strain [22,23,42]. Survival of the strain at restrictive temperature required expression of a heterologous BPL that was capable of biotin transfer to the endogenous BCCP subunit of the E. coli ACC.

The biotechnology applications of E. coli BirA depend on the apparent promiscuity in post-translational biotin addition. Schatz demonstrated, using combinatorial peptide libraries, that the E. coli enzyme is capable of catalyzing biotin transfer to short (14 residue) peptides that bear no sequence similarity to the natural substrates [43]. This peptide was shown in kinetic studies to be biotinylated, in a BirA-dependent reaction, at a rate identical with that measured for the natural substrate [44]. However, no BPL other than the E. coli enzyme shows activity towards this peptide substrate and another peptide that serves as a substrate for the S. cerevisiae BPL is not biotinylated by the E. coli enzyme [45]. Nevertheless, the activity of these peptides as BPL substrates is consistent with promiscuity in substrate recognition. Finally, the proximity biotinylation, BioID, method relies on the fact that the intermediate, biotinoyl-5′-AMP, can nonenzymatically biotinylate lysine residues. Indeed, mixing of free bio-5′-AMP with the EcBCCP results in selective biotinylation of the target lysine residue [46]. In the BioID method, biotinylation of lysine residues that are within close proximity to a BirA mutant (R118G) is achieved because the intermediate, which is readily released from the mutant, diffuses to nearby proteins to nonenzymatically biotinylate available lysine residues [47,48]. Interspecies biotinylation, the ability of BPLs to biotinylate minimal peptides and the nonenzymatic biotinylation support the notion that post-translational biotin addition is a low-specificity reaction.

Kinetic measurements of the two-step biotin transfer reaction

Steady-state measurements of post-translational biotin addition

In the two-step biotin transfer to BCCP, the biotin is first adenylated to form biotinoyl-5′-AMP. The resulting enzyme intermediate complex binds to apoBCCP to transfer biotin to the target lysine with release of AMP. Steady-state measurements of transfer are made indirectly by monitoring holocarboxylase activity [49]. Direct measurements of the overall reaction are achieved by quantifying the incorporation of radiolabeled biotin (3H or 14C) into BCCP or by measuring AMP release [36,49,50]. Measurements of the initial rates of product formation at a series of biotin, ATP or BCCP concentrations, while maintaining the other two substrates at constant concentration, yields the Michaelis constant, KM, and maximal velocity, Vmax, with respect to the varied substrate.

Single turnover measurements of the two half reactions in biotin addition

A more detailed picture of the mechanism of biotin transfer and the influence of the substrate on the process is obtained by separately measuring each half reaction [14,51]. Measurements of these ‘half reactions’, which can be rapid, are achieved using either stopped-flow or quench-flow rapid mixing techniques [16]. The first half reaction is readily monitored by changes in the intrinsic BPL fluorescence. When a solution of the biotin-bound BPL is rapidly mixed with an ATP solution the resulting bio-5′-AMP synthesis is accompanied by quenching of the intrinsic BPL fluorescence (Figure 6). Analysis of the single exponential decrease in fluorescence upon mixing yields the apparent rate of the reaction at a given ATP concentration. By repeating the measurement over a range of ATP concentrations one can obtain the Km for ATP and the maximal rate of bio-5′-AMP synthesis. This assay has been applied to the E. coli, P. horikoshii and human BPLs [14,16].

Stopped-flow fluorescence measurements of BPL-catalyzed bio-5′-AMP synthesis.

Figure 6.
Stopped-flow fluorescence measurements of BPL-catalyzed bio-5′-AMP synthesis.

(A) In measuring the ATP dependence of intermediate synthesis the BPL biotin complex is rapidly mixed with ATP. (B) Bio-5′-AMP synthesis results in quenching of the intrinsic BPL fluorescence (black spectrum:BPL.biotin, red spectrum:BPL.bio-5′-AMP). Measurements of the time-dependence of the fluorescence decrease yields the rate of bio-5′-AMP synthesis at a single ATP concentration. (C) Results of measurements of the ATP dependence of bio-5′-AMP synthesis catalyzed by the human BPL. See the text for discussion of the relationship of the curves to the kinetic mechanism of bio-5′-AMP synthesis. The figure was adapted from ref. [16].

Figure 6.
Stopped-flow fluorescence measurements of BPL-catalyzed bio-5′-AMP synthesis.

(A) In measuring the ATP dependence of intermediate synthesis the BPL biotin complex is rapidly mixed with ATP. (B) Bio-5′-AMP synthesis results in quenching of the intrinsic BPL fluorescence (black spectrum:BPL.biotin, red spectrum:BPL.bio-5′-AMP). Measurements of the time-dependence of the fluorescence decrease yields the rate of bio-5′-AMP synthesis at a single ATP concentration. (C) Results of measurements of the ATP dependence of bio-5′-AMP synthesis catalyzed by the human BPL. See the text for discussion of the relationship of the curves to the kinetic mechanism of bio-5′-AMP synthesis. The figure was adapted from ref. [16].

Results obtained for measurements of the human BPL-catalyzed first half-reaction illustrate the utility of single turnover kinetics measurements for this system. Analysis of the initial kinetic traces (Figure 6B, inset) obtained from mixing BPL:biotin with ATP indicated a two-step process with a rapid step preceding a slower step. The simplest model that adheres to these observations is shown in the following scheme:

 
formula

Measurements of the concentration-dependencies of each step yielded the rate of ATP association with BPL:biotin from the linear dependence of the apparent rate, k1, on ATP concentration (Figure 6C, inset). The dependence of the rate of the second step on ATP concentration yielded a KM for ATP of 50 ± 20 μM and a kCAT (maximum value of k2) of 0.11 ± 0.02 s−1. Comparison of these results to the steady-state measurements of the overall reaction revealed that the rate-determining step in the two-step process is synthesis of the adenylate intermediate, as indicated by the equivalence of the maximal rates for both the overall and the first half-reaction [16].

The second half-reaction, biotin transfer from the adenylated intermediate to apoBCC, can be measured using either stopped-flow or quench-flow techniques. In both techniques, the preformed BPL:bio-5′-AMP complex is rapidly mixed with excess apoBCC. The stopped-flow measurement is monitored by fluorescence since biotin transfer to BCCP returns the enzyme to its apo-state, which is characterized by a higher intrinsic fluorescence than the intermediate-bound form (Figure 7A). For the quench-flow measurement, the biotin substrate used to prepare the bio-5′-AMP intermediate is radiolabeled with 3H. Consequently, the product in the second half reaction, 3H-bioBCCP, can be quantified by scintillation counting after acid quenching and precipitation to remove excess unincorporated 3H-biotin (Figure 7B). The combined stopped-flow and quench-flow techniques allow for measurements of the second step in biotinylation from the perspective of both the enzyme:intermediate disappearance and the product appearance. Apparent rates are measured over a range of acceptor protein concentrations to obtain rate versus substrate concentration profiles.

Methods for measuring biotin transfer from bio-5′-AMP to apoBCCP.

Figure 7.
Methods for measuring biotin transfer from bio-5′-AMP to apoBCCP.

(A) BPL.bio-5′-AMP is rapidly combined with apoBCCP, which results in an increase in the intrinsic BPL fluorescence (black curve: BPL.bio-5′-AMP, red curve: BPL). The rate of biotin transfer is obtained by measuring the time-dependence of the fluorescence increase (Insert). (B) A complex of BPL.3H-bio-5′-AMP is rapidly mixed with apoBCCP and incorporation of the radiolabel into BCCP is measured following quenching of the reaction. Adapted from ref. [17].

Figure 7.
Methods for measuring biotin transfer from bio-5′-AMP to apoBCCP.

(A) BPL.bio-5′-AMP is rapidly combined with apoBCCP, which results in an increase in the intrinsic BPL fluorescence (black curve: BPL.bio-5′-AMP, red curve: BPL). The rate of biotin transfer is obtained by measuring the time-dependence of the fluorescence increase (Insert). (B) A complex of BPL.3H-bio-5′-AMP is rapidly mixed with apoBCCP and incorporation of the radiolabel into BCCP is measured following quenching of the reaction. Adapted from ref. [17].

Measurements of the rate versus concentration profiles for the second half-reaction indicate that it is limited by the rate of binding of the enzyme–intermediate complex to the substrate apoBCC. The rate versus concentration plots obtained by both stopped-flow and quench-flow indicate a linear dependence with no leveling off of the rate at a maximum value (Figure 8). This is observed both when monitoring product accumulation and disappearance of the enzyme–intermediate. The simplest interpretation of these results is that the rate depends solely on the rate of ternary complex formation between the BPL:bio-5′-AMP and the substrate BCC.

Substrate discrimination in inter-species biotin addition.

Figure 8.
Substrate discrimination in inter-species biotin addition.

Rates of PhBPL-catalyzed biotin transfer to () HsBCCP, () PhBCCP, and () EcBCCP measured by stopped-flow fluorescence and quench flow: () HsBCCP, () PhBCCP, () EcBCCP. Adapted from [17].

Figure 8.
Substrate discrimination in inter-species biotin addition.

Rates of PhBPL-catalyzed biotin transfer to () HsBCCP, () PhBCCP, and () EcBCCP measured by stopped-flow fluorescence and quench flow: () HsBCCP, () PhBCCP, () EcBCCP. Adapted from [17].

Does substrate discrimination occur in interspecies biotin addition?

As indicated previously, cross species biotinylation has been observed both in vitro and in vivo. Armed with quantitative techniques to measure the biotin transfer kinetics, we set out to assess specificity in interspecies biotin transfer. BPL enzymes and BCC substrates from archael, P. horikoshii, eubacterial, E. coli and eukaryotic, H. sapiens were used in the measurements [17].

For all combinations of enzyme and substrate that could be measured the rate versus concentration profile exhibited a linear dependence on BCC concentration with no leveling off (Figure 8). Moreover, identical dependencies were observed in monitoring enzyme:intermediate disappearance by stopped-flow fluorescence and product appearance by quantification of biotin incorporation into BCC. Thus, for all interspecies biotin transfer reactions the selectivity reflects the rate of binding of the BPL:bio-5′-AMP complex with the apoBCC substrate.

Measurements of interspecies biotin transfer indicate specificity in biotin transfer with a hierarchy of selectivity measured for the enzymes from the three classes (Table 1, [17]). The P. horikoshii enzyme exhibits the least discrimination. Indeed, this enzyme prefers the heterologous human substrate from propionyl-CoA carboxylase, over its cognate substrate. The E. coli enzyme shows intermediate discrimination and the human enzyme shows the greatest discrimination with very low or unmeasurable rates of biotin transfer to both the E. coli and P. horikoshii substrates. At the substrate level, the P. horikoshii BCC shows no detectable activity when tested against the heterologous E. coli and human enzymes. These results indicate that although interspecies biotinylation is observed in vivo, the rates of this process can vary over a broad range of magnitudes.

Table 1.
Stopped-flow measurements of biotin transfer rates for cognate and noncognate BPL–BCCP pairs
 HsBCCP1 (M−1 s−1EcBCCP1 (M−1 s−1PhBCCP1 (M−1 s−1
HsBPL 35 000 ± 3000 270 ± 40 N.D.2 
EcBPL 32 000 ± 2000 11 500 ± 700 N.D.2 
PhBPL 43 000 ± 50003 4200 ± 3003 15 000 ± 6003 
 HsBCCP1 (M−1 s−1EcBCCP1 (M−1 s−1PhBCCP1 (M−1 s−1
HsBPL 35 000 ± 3000 270 ± 40 N.D.2 
EcBPL 32 000 ± 2000 11 500 ± 700 N.D.2 
PhBPL 43 000 ± 50003 4200 ± 3003 15 000 ± 6003 
1

The errors correspond to the standard deviation of three independent stopped-flow measurements.

2

N.D., not detectable.

3

Measurements with PhBPL were carried out at 40°C, in 10 mM Tris, pH 7.5, 500 mM KCL, 2.5 mM MgCl2.

All other measurements were performed at 20°C in 10 mM Tris, pH 7.5, 200 mM KCL, 2.5 mM MgCl2. Adapted from ref. [17].

Specificity in biotin addition to human substrates

Based on the observed broad range of biotin transfer rates measured for the interspecies reaction, we set out to determine if the human enzyme shows any discrimination in biotin transfer to the five endogenous carboxylase substrates. Prior to reviewing these results it is useful to review the human biotin cycle and the metabolic contributions of biotin-dependent carboxylases.

The human (mammalian) biotin cycle in humans is complex (Figure 9A). Humans do not synthesize biotin but must obtain it from the diet [52] or, perhaps, from gut bacteria. Biotin is transported into cells via the sodium-dependent multi-vitamin transporter (SMVT) [53,54]. Once in the cell, it is incorporated into biotin-dependent carboxylases in the two-step reaction catalyzed by the human BPL, which is referred to as holocarboxylase synthetase or HCS in mammals. Turnover of the carboxylase proteins yields biotin either bound to free lysine as biocytin (biotinoyl-lysine) or in short peptides. The biotin is released for recycling by the enzyme biotinidase, which catalyzes hydrolysis of the amide linkage [55].

Mammalian biotin cycle and metabolic roles of biotin-dependent carboxylases.

Figure 9.
Mammalian biotin cycle and metabolic roles of biotin-dependent carboxylases.

(A) Biotin is transported into cells via the SMVT and then linked to the five biotin-dependent carboxylases. Following protein turnover the biotin is recycled by biotinidase-catalyzed hydrolysis. (B) Biotin-dependent carboxylases function in gluconeogenesis (PC), fatty acid synthesis and breakdown (ACC1 and 2), leucine degradation (β-methyl crotonyl-CoA carboxylase: MCC) and odd chain fatty acid degradation (PCC).

Figure 9.
Mammalian biotin cycle and metabolic roles of biotin-dependent carboxylases.

(A) Biotin is transported into cells via the SMVT and then linked to the five biotin-dependent carboxylases. Following protein turnover the biotin is recycled by biotinidase-catalyzed hydrolysis. (B) Biotin-dependent carboxylases function in gluconeogenesis (PC), fatty acid synthesis and breakdown (ACC1 and 2), leucine degradation (β-methyl crotonyl-CoA carboxylase: MCC) and odd chain fatty acid degradation (PCC).

Human BCs contribute to several critical metabolic processes, including gluconeogenesis, fatty acid synthesis and breakdown and amino acid catabolism (Figure 9B). Mutations in the genes that code for HCS or biotinidase can result in either early or late onset multiple carboxylase deficiency (MCD), respectively [56,57]. Cases have been reported in which late onset disease is linked to mutations in the gene that codes for HCS [58]. Newborn screening for both HCS [59] and biotinidase [60] linked MCD is now available and, once diagnosed, most symptoms are successfully alleviated by administration of biotin in high doses.

In assessing the activities of the eukaryotic carboxylases in post-translational biotin addition it is important to consider their subcellular localization. The five human biotin-dependent carboxylases include ACC 1 and 2, PC, MCC and PCC. While ACC1 is a cytosolic enzyme, ACC2 is associated with mitochondrial outer membrane on the cytosolic side [61]. PC, MCC and PCC are all located in the mitochondria matrix [6264]. Since all of the enzymes are encoded in nuclear, not mitochondrial, DNA, translocation to the mitochondrion must occur post-translationally. Whether the biotin addition occurs pre- or post-mitochondrial localization remains to be determined. Studies of the HCS localization indicate that it is primarily in the cytosol [65,66]. However, data showing that PC could be labeled with 35S-biotin both pre- and post-mitochondrial translocation supports the presence of HCS activity in the mitochondrion [67].

Human HCS exhibits selectivity in post-translational biotin addition

Selectivity in human HCS-catalyzed biotin transfer to the five endogenous substrates was investigated using the kinetic assays previously described [68]. The BCC domains derived from the five carboxylase parent enzymes were used as substrates in the measurements. Both the full-length HCS and a splice variant that is initiated at residue 58 of the enzyme were used for the measurements. The second half-reaction, biotin transfer from bio-5′-AMP to the acceptor protein, was measured using both the stopped-flow and quench-flow assays described above.

Rate measurements at a single BCC concentration yielded single exponential kinetics for all substrates. For the mitochondrial substrates, the rate versus concentration profiles yielded linear dependencies and the apparent bimolecular rate of association of the enzyme intermediate with the substrate acceptor protein was obtained from linear regression of the data (Figure 10, [68]). The results revealed a 3.5-fold range of rates for the three substrates with the order from fastest to slowest being PCC > MCC > PC (Table 2, [68]).

Human BPL (HCS) distinguishes among carboxylase substrates in post-translational biotin addition.

Figure 10.
Human BPL (HCS) distinguishes among carboxylase substrates in post-translational biotin addition.

Results of quench-flow measurements of HCS-catalyzed biotin addition to the BCCP segments of PCC(o) and ACC1(m). The lines represent the results of linear (PCC) and nonlinear (ACC1) regression of the rate versus [BCCP] data, with the ACC1 results shown in the insert. The figure was adapted from data presented in [68].

Figure 10.
Human BPL (HCS) distinguishes among carboxylase substrates in post-translational biotin addition.

Results of quench-flow measurements of HCS-catalyzed biotin addition to the BCCP segments of PCC(o) and ACC1(m). The lines represent the results of linear (PCC) and nonlinear (ACC1) regression of the rate versus [BCCP] data, with the ACC1 results shown in the insert. The figure was adapted from data presented in [68].

Table 2.
HCS-catalyzed biotin transfer rates to PCC, MCC and PC BCCP
BCCP Stopped flow1 (×104 M−1 s−1Quench Flow2 (×104 M−1 s−1
PCC 3.5 ± 0.32 3.1 ± 0.3 
MCC 1.9 ± 0.2 1.9 ± 0.2 
PC 1.00 ± 0.07 1.00 ± 0.05 
BCCP Stopped flow1 (×104 M−1 s−1Quench Flow2 (×104 M−1 s−1
PCC 3.5 ± 0.32 3.1 ± 0.3 
MCC 1.9 ± 0.2 1.9 ± 0.2 
PC 1.00 ± 0.07 1.00 ± 0.05 
1,2

The errors represent the standard deviation of the rates obtained from three independent stopped-flow or two independent quench-flow measurements. Adapted from ref. [68].

The two ACC substrates displayed kinetic behavior in HCS-catalyzed biotin addition strikingly distinct from that observed for the mitochondrial substrates (Figure 10, [68]). First, the biotin transfer rates at a given BCC concentration were consistently much lower (100×) for the two ACC substrates relative to the PCC, PC and MCC substrates. Second, in contrast with the linear dependencies of rates on BCC concentration observed for the mitochondrial substrates, the apparent rates showed a hyperbolic dependence on substrate BCC concentration (Figure 10, inset). This hyperbolic dependence was analyzed using a Michaelis–Menten model to obtain the KM and kCAT values for the reaction. The magnitude of the kCAT value for ACC2 indicates that for this substrate the second step in biotin transfer, not the first step, is rate limiting for the overall reaction (Table 3).

Table 3.
HCS-catalyzed biotin transfer to ACC1 and ACC2 BCCP
 Stopped flow 3H-biotin incorporation 
KM (µM) kcat (s−1KM (µM) kcat (s−1
ACC2-his6 80 ± 201 0.032 ± 0.0051 60 ± 202 0.037 ± 0.0042 
ACC1-his6 190 ± 903 0.12 ± 0.043 70 ± 302 0.06 ± 0.022 
 Stopped flow 3H-biotin incorporation 
KM (µM) kcat (s−1KM (µM) kcat (s−1
ACC2-his6 80 ± 201 0.032 ± 0.0051 60 ± 202 0.037 ± 0.0042 
ACC1-his6 190 ± 903 0.12 ± 0.043 70 ± 302 0.06 ± 0.022 
1

The errors represent the standard deviation of the rates obtained in three independent stopped-flow experiments, or the propagated 68% confidence interval obtained from the individual fits, whichever was larger.

2

The errors correspond to the standard deviation of results obtained in two independent experiments, or the propagated 68% confidence interval obtained from the individual fits, whichever was larger.

3

The errors represent the propagated 68% confidence limits obtained from globally fitting the data, which comprised three independent datasets for the three lower concentrations, and at least 10 traces for the three highest concentrations. Adapted from [68].

Origins of the apparent lack of specificity in post-translational biotin addition

The results described above contradict previous reports of nonspecificity in post-translational biotin addition [27,40]. The discrepancy is likely due to the different methods used to measure the addition. For in vivo observations the measurements were qualitative, involving either BCCP-mediated derepression of transcription regulation by E. coli BirA or observation of a band in gels corresponding to the biotinylated product [27]. Even for kinetic measurements of interspecies biotinylation, the measurements were of the overall two-step reaction. Given our observation that the rate-limiting step is the synthesis of bio-5′-AMP from biotin and ATP, steady-state kinetic measurements of the overall reaction are incapable of detecting selectivity in biotin transfer to the BCCP.

In light of the observed selectivity in the second half reaction it would be informative to reexamine the reactivities of the isolated BCC segments relative to the full biotin-dependent carboxylase. We previously showed that the intact BCCP subunit of E. coli Acetyl-CoA carboxylase is equivalent to the isolated BCC domain in biotin transfer [51] in stopped-flow fluorescence transfer measurements. However, we do not know if this result is universally true. The availability of intact S. cerevisiae ACC [11] and S. aureus PC [69] as well as their cognate BPLs [22,70,71] provides an opportunity to test the equivalence between the intact and minimal substrates in biotin transfer using the single turnover techniques developed in our laboratory.

The physiological significance of the specificity in HCS-catalyzed biotin addition to the five carboxylase substrates

Although the differences in rates of biotin transfer to the five human carboxylase substrates are striking, its physiological significance remains unclear. The mitochondrial substrates are biotinylated more rapidly than the cytosolic, and it is possible that this reflects the necessity of modifying these substrates prior to their translocation to the organelle. However, although the majority of the human BPL is found in the cytoplasm [65,66], there is some evidence that mitochondrial carboxylases can be biotinylated after translocation [67]. A detailed examination of HCS localization would prove useful in understanding the relationship between the kinetics of post-translational biotin addition to the mammalian carboxylases and their subcellular localization.

The different rates of biotin transfer to the carboxylases may be significant for biotin distribution, particularly in conditions of limiting biotin. In biotin deficient conditions, the activities of biotin-dependent carboxylases are decreased but can be rapidly restored by biotin addition and does not require new protein synthesis [72,73]. Rather, only the proportion of the apo- and holo-forms of the carboxylases changes. The differences in the intrinsic rates of biotin transfer to the five mammalian carboxylases may play a role in regulating the activities of these enzymes, particularly under conditions of biotin limitation.

Structural origins of selectivity in post-translational biotin addition

The selectivity in post-translational biotin addition must have a structural basis. It has previously been suggested that the N-terminus of the human BPL (HCS) functions in substrate discrimination [32]. This is consistent with the high selectivity of this enzyme toward its cognate substrate observed in the interspecies biotin transfer measurements (Table 1). Moreover, several mutations in the hcs gene associated with Multiple Carboxylase Deficiency result in amino acid substitutions in the N-terminal region of the protein [21]. Future structural and biochemical investigations will reveal the molecular basis of the contribution of the HCS N-terminal domain to post-translational biotin addition to mammalian carboxylases.

Abbreviations

     
  • ACC

    acetyl-CoA carboxylase

  •  
  • BC

    biotin carboxylase

  •  
  • BCC

    biotin carboxyl carrier

  •  
  • BPLs

    biotin protein ligases

  •  
  • CT

    carboxyl transferase

  •  
  • MCC

    methylcrotonyl-CoA carboxylase

  •  
  • PC

    pyruvate carboxylase

  •  
  • PCC

    propionyl-CoA carboxylase

  •  
  • SMVT

    sodium-dependent multi-vitamin transporter

Competing Interests

The Author declares that there are no competing interests associated with this manuscript.

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