Biotin, carboxylations with

However, if we can design some sophisticated routes to generate carbanion equivalents in the active site of the enzyme, carboxylation reaction might be possible. In fact, acetyl-CoA is carboxylated with the aid of biotin in the biosynthetic pathway of long-chain fatty acids. 

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. ... 

A last example of a postfunctionalized PPE was reported by Bunz et al. A grafted PPE made by polymerization of a macromonomer was treated with a biotin carboxylic acid chloride in the absence or in the presence of triethy-... 

Biotin (5) is the coenzyme of the carboxylases. Like pyridoxal phosphate, it has an amide-type bond via the carboxyl group with a lysine residue of the carboxylase. This bond is catalyzed by a specific enzyme. Using ATP, biotin reacts with hydrogen carbonate (HCOa ) to form N-carboxybiotin. From this activated form, carbon dioxide (CO2) is then transferred to other molecules, into which a carboxyl group is introduced in this way. Examples of biotindependent reactions of this type include the formation of oxaloacetic acid from pyruvate (see p. 154) and the synthesis of malonyl-CoA from acetyl-CoA (see p. 162). 

A third member of the bimolecular then unimolecular reaction class is a variant of the previous mechanism. In this case, the conjugate base of biotin reacts with bicarbonate to produce an addition intermediate that then reacts with ATP (Scheme 23). It is likely that the phosphorus of the terminal group of ATP would preassociate with an oxygen of bicarbonate. In particular, if the anionic center of bicarbonate associates with a cation, the 7r-electron density of bicarbonate would align with the phosphorus of the terminal phosphate of ATP. The addition of the conjugate base of a urea to a carboxylate is an appropriate model for this mechanism. The intermediate should be very reactive toward ATP based on the observation that the conjugate base of a carbonyl hydrate reacts rapidly with an internal phosphate ester (59). 

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. 

Carboxylation reactions occur in the biosynthesis of fatty acids, in the degradation of leucine and isoleucine, and in the degradation of fatty acids with uneven numbers of C atoms. Acetyl-CoA carboxylase (EC 6.4.1.2) catalyses the following reaction in the biosynthesis of fatty acids Acetyl-CoA + HC03 + H + ATP —> malonyl-CoA + ADP + Pj. The monomer of this enzyme is composed of 4 different subunits. One of these, biotin carboxylase (III), catalyses the carboxylation of the biotin residue. This biotin residue is covalently bound to the second subunit, which is known as the biotin-carboxyl carrier protein (biotin-CCP) Biotin-CCP + HCOj + H + ATP ->... 

Then the 7,8-diaminononanoate (7,8-diaminopelargonic acid) (Scheme 12.93) is converted to the bisamide dethiobiotin (an imidazolidone) by carboxylation with carbon dioxide (CO2) in the presence of ATP in a reaction requiring magnesium (Mg+ ) and catalyzed by dethiobiotin synthase (EC 6.3.3.3). The well-known reversible reaction of amines with carbon dioxide (CO2) and an X-ray crystal structure showing the 8-amino group as a mixed carbonic phosphate anhydride has lent support to this picture (PDB la82). Finally, in the presence of biotin synthase (EC 2.8.1.6), an enzyme that appears to be used only once in each synthesis, reaction occurs with 2 equivalents (but almost certainly one at a time) of SAM and sulfur to produce biotin, 2 equivalents of methionine (Met, M), and 2 equivalents of 5 -deoxyadenosine. 

Fig 1 Plasmid maps of the three expression plasmids used to express Biotin Carboxyl Carrier Protein without transit peptide, BCCP with transit peptide and Biotin Carboxylase without transit peptide. 

We have overexpressed the B. napus biotin carboxyl carrier protein and biotin carboxylase. Whilst most of the protein is in an insoluble form, a fraction of the BCCP is present as soluble protein and this may or may not be complexed with the biotin carboxylase. Initial gel permeation experiments give inconclusive results, in that no substantial difference in flow through the column is seen if BC is expressed in the bacteria with BCCP or not. On the other hand, the initial elution fraction for the overexpressed BCCP equates to a molecular weight of some 60-70kDa, the same fraction as the E. coli BCCP elutes in. Clearly further experimentation is required to asses the degree of interaction and what proteins BCCP will interact with. 

Acetyl-CoA carboxylase (ACCase) catalyses the ATP-dependant carboxylation of acetyl-CoA to form malonyl-CoA, thus providing the essential substrate for fatty acid biosynthesis. Dicotyledonous plants contain two forms of ACCase a multifunctional enzyme (typel) wich is presumed to be cytosolic, and a multi-subunit complex (typell) located in the plastid wich is responsible for de novo fatty acid synthesis. In prokaryotes, the ACCase is a type II enzyme comprising biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP) and a carboxyl transferase with two subunits (CTa and CTp). The cDNA encoding the B.napus CXp and BCCP have already been cloned (Elborough et a/.,1995) as have the cDNAs encoding the BC and BCCP from tobacco and Arabidopsis respectively (Shorrosh et al, 1995 Choi et al.y 1995). 

Acetyl-CoA carboxylase (ACCase) catalyzes the ATP dependent carboxylation of acetyl-CoA to form malonyl-CoA. This reaction is the first committed step in fatty acid biosynthesis and is a potentially rate-limiting step in the process. Two forms of ACCase have been observed - a prokaryotic and a eukaryotic form. In the prokaryotic form, the biotin carboxylase, biotin carboxyl carrier protein and carboxyl transferase components of ACCase are associated with different polypeptides in a multi-subunit (MS) complex (Samols et al. 1988). In contrast, the eukaryotic form comprises multimers of a single multifunctional (MF) polypeptide of200 kDa. Dicots have been reported to have an MS plastidial ACCase and an MF extra-plastidial ACCase while grasses and other monocots are believed to have the MF ACCase in both plastids and extra-plastidial sources (Sasaki et al. 1995). 

Neither catalytic component contains a trace of bound biotin. The biotin prosthetic group is covalently linked to the third component, carboxyl carrier protein (CCP—biotin). As with other biotin enzymes, the bicyclic ring of the prosthetic group resides at the distal end of a flexible 14 A side chain which allows it to act as a mobile carboxyl carrier between the two catalytic centers as illustrated below. A large number of biotin-dependent enzymes which carry out diverse reaction types are now known. All of these reactions proceed through a carboxylated intermediate with the carboxybiotinyl prosthetic group functioning as a mobile carboxyl carrier . 

Two other reactions for succinyl C5oA synthesis may be noted. In heart muscle a CoA transferase is present that requires guanosine triphosphate (GTP) or inosine triphosphate (ITP) but not ATP itaconate can replace succinate in this reaction (54, In animals and bacteria a CoA transferase is found that transfers CoA from acetoacetyl CoA to succinate the enzyme in liver is not highly active. It is thought that in propionic bacteria propionyl CoA is carboxylated with a biotin enzyme to methylmalonyl CoA the reversible conversion of methylmalonyl CoA to succinyl CoA is catalyzed by a Bu enzyme (54a). 

The carboxylation reactions of the carboxylase enzyme are also inhibited by avidin, specific biotin-binding protein from egg white. Experimental feeding of avidin or raw egg white results in biotin deficiency with features of combined carboxylase deficiency, since the avidin-biotin complex is not absorbed from the gut. Avidin has four biotin-binding sites and also inhibits biotin-containing enzymes by effectively decarboxylating and preventing re-carboxylation of the biotin moiety (Moss and Lane, 1971). 

Fatty acids with odd numbers of carbon atoms are rare in mammals, but fairly common in plants and marine organisms. Humans and animals whose diets include these food sources metabolize odd-carbon fatty acids via the /3-oxida-tion pathway. The final product of /3-oxidation in this case is the 3-carbon pro-pionyl-CoA instead of acetyl-CoA. Three specialized enzymes then carry out the reactions that convert propionyl-CoA to succinyl-CoA, a TCA cycle intermediate. (Because propionyl-CoA is a degradation product of methionine, valine, and isoleucine, this sequence of reactions is also important in amino acid catabolism, as we shall see in Chapter 26.) The pathway involves an initial carboxylation at the a-carbon of propionyl-CoA to produce D-methylmalonyl-CoA (Figure 24.19). The reaction is catalyzed by a biotin-dependent enzyme, propionyl-CoA carboxylase. The mechanism involves ATP-driven carboxylation of biotin at Nj, followed by nucleophilic attack by the a-carbanion of propi-onyl-CoA in a stereo-specific manner. 

---