Pendent homodimer of 41.6 kDa of known crystal structure (34, 35). The enzyme condenses alanine with pimeloyl-CoA to give 7-keto-8-amino pelargonic acid (formal name, 8-amino-7oxononanoic acid) plus CoA and CO2 (resulting from decarboxylation of alanine). BioF is a two-domain protein with the pyridoxal phosphate bound in a crevice between the two domains formed by residues of both domains. The mechanism of the enzyme has been studied in some detail (36). Historically the enzyme has been assayed using pimeloyl-CoA although pimeloyl-ACP could be the physiological substrate in E. coli (ACP-requiring enzymes will often use the analogous CoA compound as a model substrate). Consistent with this notion the E. coli 7-keto-8-amino pelargonic acid synthase has a much higher Michaelis constant for pimeloyl-CoA than the analogous enzyme from Bacillus sphaericus (37), an organism in which pimeloyl-CoA is thought to be the physiological substrate due to the presence of pimeloyl-CoA synthetase. BioA BioA is 7,8-diaminopelargonic acid (DAPA) aminotransferase (the formal name of DAPA is 7,8-diaminononanoate) that has many similarities to BioF, the preceding enzyme in the pathway. Although the BioA subunit (47.3 kDa) is slightly larger than that of BioF, it is also a homodimeric pyridoxal phosphate-dependent enzyme. Indeed, the overall structure of BioA is very similar to that of BioF (38) and this is reflected in a weak sequence homology. BioA is a transaminase that converts KAPA to DAPA and as such is not a particularly interesting enzyme (39, 40). However, the amino donor is not a standard amino acid, but rather the highly activated amino acid SAM (39, 41) which requires three ATP equivalents for its VelpatasvirMedChemExpress GS-5816 synthesis. The deaminated product derived from SAM, S-adenosyl-2-oxo-4thiomethylbutryate, spontaneously degrades in vitro (39), and thus it seems likely that three ATP equivalents are consumed in what is an otherwise simple transamination reaction. The expense of this Crotaline site perplexing choice of amino donor may provide a rationale for the known tight regulation of biotin synthesis. However, it could be argued that use of a more pedestrian amino donor (B. subtilis uses lysine (42)) could alleviate the need for tight regulation. BioD In contrast to the preceding enzymes BioD (dethiobiotin synthase or DTBS) catalyzes an unusually interesting step, the formation of the ureido moiety of biotin (43, 44). The BioD reaction is the ATP-dependent formation of dethiobiotin from DAPA and CO2. The enzyme is a homodimeric protein (subunit of 24.1 kDa) that is structured into a single well foldedAuthor Manuscript Author Manuscript Author Manuscript Author ManuscriptEcoSal Plus. Author manuscript; available in PMC 2015 January 06.CronanPagedomain (45?8). X-ray crystallographic studies have shown that the reaction proceeds by carbamoylation of N-7 of DAPA (45, 46) (Fig. 3). Independent NMR evidence for carbamate formation has also been obtained (49). The second partial reaction is also unusual. In this reaction the carbamate is activated by transfer of the -phosphoryl moiety of ATP to a carbamate oxygen to form a mixed anhydride (Fig. 3). This mixed anhydride species has been demonstrated by time-resolved crystallography (50). The final step of the dethiobiotin synthase reaction is a nucleophilic attack by the N-8 nitrogen of DAPA on a carbamoyl oxygen of the mixed anhydride (Fig. 3). This results in release of the phosphate group and formation of the ureido ring of dethiobiotin.Pendent homodimer of 41.6 kDa of known crystal structure (34, 35). The enzyme condenses alanine with pimeloyl-CoA to give 7-keto-8-amino pelargonic acid (formal name, 8-amino-7oxononanoic acid) plus CoA and CO2 (resulting from decarboxylation of alanine). BioF is a two-domain protein with the pyridoxal phosphate bound in a crevice between the two domains formed by residues of both domains. The mechanism of the enzyme has been studied in some detail (36). Historically the enzyme has been assayed using pimeloyl-CoA although pimeloyl-ACP could be the physiological substrate in E. coli (ACP-requiring enzymes will often use the analogous CoA compound as a model substrate). Consistent with this notion the E. coli 7-keto-8-amino pelargonic acid synthase has a much higher Michaelis constant for pimeloyl-CoA than the analogous enzyme from Bacillus sphaericus (37), an organism in which pimeloyl-CoA is thought to be the physiological substrate due to the presence of pimeloyl-CoA synthetase. BioA BioA is 7,8-diaminopelargonic acid (DAPA) aminotransferase (the formal name of DAPA is 7,8-diaminononanoate) that has many similarities to BioF, the preceding enzyme in the pathway. Although the BioA subunit (47.3 kDa) is slightly larger than that of BioF, it is also a homodimeric pyridoxal phosphate-dependent enzyme. Indeed, the overall structure of BioA is very similar to that of BioF (38) and this is reflected in a weak sequence homology. BioA is a transaminase that converts KAPA to DAPA and as such is not a particularly interesting enzyme (39, 40). However, the amino donor is not a standard amino acid, but rather the highly activated amino acid SAM (39, 41) which requires three ATP equivalents for its synthesis. The deaminated product derived from SAM, S-adenosyl-2-oxo-4thiomethylbutryate, spontaneously degrades in vitro (39), and thus it seems likely that three ATP equivalents are consumed in what is an otherwise simple transamination reaction. The expense of this perplexing choice of amino donor may provide a rationale for the known tight regulation of biotin synthesis. However, it could be argued that use of a more pedestrian amino donor (B. subtilis uses lysine (42)) could alleviate the need for tight regulation. BioD In contrast to the preceding enzymes BioD (dethiobiotin synthase or DTBS) catalyzes an unusually interesting step, the formation of the ureido moiety of biotin (43, 44). The BioD reaction is the ATP-dependent formation of dethiobiotin from DAPA and CO2. The enzyme is a homodimeric protein (subunit of 24.1 kDa) that is structured into a single well foldedAuthor Manuscript Author Manuscript Author Manuscript Author ManuscriptEcoSal Plus. Author manuscript; available in PMC 2015 January 06.CronanPagedomain (45?8). X-ray crystallographic studies have shown that the reaction proceeds by carbamoylation of N-7 of DAPA (45, 46) (Fig. 3). Independent NMR evidence for carbamate formation has also been obtained (49). The second partial reaction is also unusual. In this reaction the carbamate is activated by transfer of the -phosphoryl moiety of ATP to a carbamate oxygen to form a mixed anhydride (Fig. 3). This mixed anhydride species has been demonstrated by time-resolved crystallography (50). The final step of the dethiobiotin synthase reaction is a nucleophilic attack by the N-8 nitrogen of DAPA on a carbamoyl oxygen of the mixed anhydride (Fig. 3). This results in release of the phosphate group and formation of the ureido ring of dethiobiotin.