Carboxylases biotinylation

Animal and fungal ACCs are comprised of large multifunctional polypeptides containing the biotin carboxylase, biotinyl carboxyl carrier protein, and carboxyltransferase... 

Protein biotinylation is catalyzed by biotin protein ligase (BPL). In the active site of the enzyme, biotin is activated at the expense of ATP to form AMP-biotin the activated biotin can then react with a nucleophile on the targeted protein. BPL transfers the biotin to a special lysine on biotin carboxyl carrier protein (BCCP), a subunit of AcCoA carboxylase (Scheme 21). Biotinylation of BCCP is very important in fatty acid biosynthesis, starting the growth of the fatty acid with AcCoA carboxylase to generate malonyl-CoA. Recently the crystal structures of mutated BPL and BCCP have been solved together with biotin and ATP to get a better idea of how the transfer fiinctions. ... 

This enzyme [EC 4.1.1.41 ], also known as propionyl-CoA carboxylase, catalyzes the conversion of (5)-2-methyl-3-oxopropanoyl-CoA to propanoyl-CoA and carbon dioxide. The enzyme from Veillondla alcalescens is a biotinyl-protein, requires sodium ions, and acts as a sodium pump. 

MECHANISM FIGURE 16-16 The role of biotin in the reaction catalyzed by pyruvate carboxylase. Biotin is attached to the enzyme through an amide bond with the e-amino group of a Lys residue, forming biotinyl-enzyme. Biotin-mediated carboxylation reactions occur in two phases, generally catalyzed by separate active sites on the enzyme as exemplified by the pyruvate carboxylase reaction. In the first phase (steps to ), bicarbonate is converted to the more activated C02, and then used to carboxylate biotin. The bicarbonate is first activated by reaction with ATP to form carboxyphosphate (step ), which breaks down to carbon dioxide (step ). In effect, the... 

Structures of biotinyl enzyme and N -carboxybiotin. The reactive portions of the coenzyme and the active intermediate are shown in red. In carboxylase enzymes, biotin is covalently bonded to the proteins by an amide linkage between its carboxyl group and a lysyl- -NH2 group in the polypeptide chain. 

The coenzymatic function of biotin appears to be to mediate the carboxylation of substrates by accepting the ATP-activated carboxyl group and transferring it to the carboxyl acceptor substrate. There is good reason to believe that the enzymatic sites of ATP-dependent carboxylation of biotin are physically separated from the sites at which N -carboxybiotin transfers the carboxyl group to acceptor substrates, that is, the transcarboxylase sites. In fact, in the case of the acetyl-CoA carboxylase from E. coli (see chapter 18), these two sites reside on two different subunits, while the biotinyl group is bonded to a third, a small subunit designated biotin carboxyl carrier protein. 

Figure 12-4. The biotin cycle shows the actions of biotin holocarboxylase synthetase in biotinylating carboxylases and of biotinidase in cleaving biocytin, thereby recycling biotin.

Mammalian pyruvate carboxylase has four identical subunits, and the isolated monomer will catalyze the complete reaction. By contrast, three distinct subunits can be isolated from acetyl CoA carboxylase of Escherichia coli and spinach chloroplasts a biotinyl carrier protein, biotin carboxylase, and carboxyl transferase. 

The important function of biotin is its role as coenzyme for carboxylase, which catalyses carbon dioxide fixation or carboxylation reaction. The epsilon amino group of lysine in carboxylase enzymes combines with the carboxyl group of biotin to form covalently linked biotinyl carboxyl carrier protein (BCCP or biocytin) (Figure 6.8). This serves as an intermediate carrier of carbon dioxide. The carboxylation of acetyl CoA to malonyl CoA in presence of acetyl CoA carboxylase requires biotin as coenzyme. Propionyl carboxylase and pyruvate carboxylase are also associated with biotin. 

Acetyl-CoA carboxylase is a biotin-dependent enzyme. It has been purified from microorganisms, yeast, plants, and animals. In animal cells, it exists as an inactive pro-tomer (M.W. 400,000) and as an active polymer (M.W. 4-8 million). The protomer contains the activity of biotin carboxylase, biotin carboxyl carrier protein (BCCP), transcarboxylase, and a regulatory allosteric site. Each protomer contains a biotinyl group bound in amide linkage to the e-amino group of a lysyl residue. 

Proteolysis of biotin-containing enzymes releases -biotinyllysine, or biocytin. Biotinidase cleaves biocytin and biotinylated peptides, resulting from degradation of endogenous carboxylases, to biotin and lysine. Thus,... 

All four carboxylases use bicarbonate as their one-carbon substrate and, in all, the biotin is covalently linked by an amide bond between the carboxyl of biotin and an epsilon amino group of a lysyl residue in the holocarboxylase synthase (= biotin ligase) that catalyzes the formation of the covalent bond. Biotinylation of histones is involved in regulation of gene transcription and may also play a role in packaging of deoxyribonucleic acid (DNA). Biotin has also been found to inhibit the generation of reactive oxygen species (ROS) by neutrophils in vitro. 

The question of the in vivo situation was of course open. As most organisms require only tiny amounts of biotin, the hypothesis that BS could also be noncatalytic in vivo had to, and has been considered. It looks, however, more reasonable to expect a catalytic function. It is now well established that in vivo synthesis of [Fe-S] centers makes use of a very complex machinery, namely, the Isc or Suf systems in E. colt They include chaperone proteins, which may be necessary for repairing the cluster, or for the regeneration of a native empty [2Fe-2S] site. A first answer to this puzzling question has been given in a recent paper that describes in vivo experiments. The amount of biotin produced from DTB was determined by coexpression in E. coli of BS, biotin ligase, and a fragment of acetyl-CoA carboxylase to trap the biotin produced, followed by quantification of the biotinylated protein. A turnover of 20-60 equivalents of biotin has been observed, but a quantitative evaluation is difficult due to the fact that turnover renders the protein susceptible to proteolysis. 

Numerous studies performed with E. coli have established that, in E. coli, biotin regulates very efficiently its biosynthetic pathway, with an absolute specificity, the biotin vitamers being inactive. As the topics has been largely reviewed, it will be only summarized here. The regulation occurs at the transcriptional level and the biotin operon repressor (BirA) has been well characterized. This 33.5 kDa bifiinctional protein is both an enzyme and a transcriptional regulator (Figure 21). It activates biotin into biotinyl-5 -AMP with ATP (reaction a) and transfers biotin on a specific lysine residue of the biotin accepting proteins (in E. coli, the biotin carboxyl carrier protein (BCCP), a subunit of acetyl-CoA carboxylase) (reaction b). When all the... 

Each ACC half-reaction is catalyzed by a different protein sub-complex. The vitamin biotin is covalently coupled through an amide bond to a lysine residue on biotin carboxyl carrier protein (BCCP, a homodimer of 16.7-kDa monomers encoded by accB) by a specific enzyme, biotin-apoprotein ligase (encoded by birA), and is essential to activity. The crystal and solution structures of the biotinyl domain of BCCP have been determined, and reveal a unique thumb required for activity (J. Cronan, 2001). Carboxylation of biotin is catalyzed by biotin carboxylase (encoded by accC), a homodimeric enzyme composed of 55-kDa subunits that is copurified complexed with BCCP. The accB and accC genes form an operon. The three-dimensional structure of the biotin carboxylase subunit has been solved by X-ray diffraction revealing an ATP-grasp motif for nucleotide binding. The mechanism of biotin carboxylation involves the reaction of ATP and CO2 to form the shortlived carboxyphosphate, which then interacts with biotin on BCCP for CO2 transfer to the I -nitrogen. 

Repressors may have similar recognition domains but may vary greatly both in size and in the functioning of fheir other domains, which may react both with small allosteric effectors and with other proteins. The repressor BirA of the E. coli biotin synthesis operon is an enzyme. The 321-residue protein activates biohn to form biotinyl 5 -adenylafe and transfers the biotinyl group to proteins such as acetyl-CoA carboxylase ° ° and also represses transcriphon. 

Brocklehurst, S.M. and Perham, R.N. (1993) Prediction of the three-dimensional structures of the biotinylated domain from yeast pyruvate carboxylase and of the lipoylated H-protein from the pea leaf glycine cleavage system a new automated method for the prediction of protein tertiary structure. Protein Sci. 2 626-639. 

FIGURE 18.8 The two stages of the pyruvate carboxylase reaction. COj is attached to the biotinylated enzyme. CO2 is transferred from the biotinylated enzyme to pyruvate, forming oxaloacetate. ATP is required in the first part of the reaction. 

Acetyl-CoA-carboxylase (ACCase) plays a fundamental role in fatty acid metabolism and is a biotinylated errzyme that catalyzes the carboxylation of acetyl-CoA. 

The studies of Reed and co-workers on the nature of protein-bound lipoic acid and its enzymatic release and reincorporation may be applicable to biotin-containing enzymes. It is pertinent to note that a conjugated form of biotin, biocytin, has been isolated from yeast autolyzate and identified as A -biotinyl-L-lysine (Wright et ah, 19r)2 Peck et al., 1952). Biotin is now known to be the prosthetic group of several carboxylases (see Ochoa and Kaziro, 1961, for a review of these enzymes). Although the nature of the moiety to which biotin is bound has not been established, it seems highly probable that it is the -amino group of a lysine residue. 

These enzymes are large molecules comprising three or more subunits a biotin carboxylase subunit (a-subunit) where the enzymes pickup the carboxyl group from substrates a decarboxylase subunit (p-subunit) where the decarboxylation takes place and the sodium ion pump is located and a biotinyl subunit (y-subunit) containing the specific lysine residue to which biotin covalently binds. A fourth subunit (8-subunit) has been reported in all glutaconyl-CoA decarboxylases to anchor the a-subunit to the... 

Biotin enzymes carboxylases that use biotin as a cofactor. The biotin is bound via an amide bond to the E-amino group of a specific lysine residue in the enzyme protein, i.e. B.e. contain a biotinyllysyl residue. Free (+)-E-V-biotinyl-L-lysine actually occurs in yeast extract, and is known as biocytin. During catalysis, N-atom 1 of the biotin residue is carboxylated in an ATP-dependent reaction ATP + HCOj" + bioti-nyl-enzyme (I) -> ADP + Pj + carboxybiotinyl-en-zyme (II). The carboxyl group is then transferred from (II) to the carboxylase substrate (II) + substrate —> (I) + carboxylated substrate. 

Another example of a carboxylation reaction is the formation of oxaloacetate from pyruvate. Pyruvate carboxylase (EC 6.4.1.1) consists of 4 subunits, each covalently bound to one molecule of biotin and containing one Mg ion 1. Biotinyl-enzyme + ATP + CO2 + HjO — carboxybiotinyl-enzyme + ADP + Pj 2. Carboxybiotinyl-enzyme + pyruvate biotinyl-enzyme + oxaloacetate. In the degradation of fatty acids with odd numbers of C atoms, carboxylation of pro-pionyl-CoA to methylmalonyl-CoA is also catalysed by B. Carboxybiotinyl-enzyme + CHj-CHj-CO -SCoA biotinyl-enzyme + CHj-CH(COOH)-CO -SCoA. The same reaction occurs in the degradation of isoleucine, leucine and valine. 

N ( l -N-carboxy-(+) - biotinyl )-L-lysyl enzyme Fig. 4. Carboxylated biotinyl prosthetic group of acetyl-CoA carboxylase. 

Treatment of the biocytin derivative with biotini-dase, an enzyme that splits the bond between biotin and lysine, yields l -N-methoxy-Ci4-carbonylbiotin. This mode of attachment of CO2 to biotin (in the L-N position) and of biotin to the apoenzyme (6-N-(+) biotinyl lysyl) proved to be identical for j8-methyl-crotonyl CoA carboxylase, propionyl CoA carboxyyl-ase, and oxaloacetic transcarboxylase. The binding of CO2 with biotin is a high-energy binding ( — 4.7 kilocalories per mole) and the CO2 bond to the L-N-meth-oxy-Ci4-carbonylbiotin can be considered as a form of activated CO2. 

Athappilly FK, Hendrickson WA (1995) Structure of the biotinyl domain of acetyl-coenzymeA carboxylase determined by MAD phasing. Structure 3 1407-19... 

---