Dethiobiotin Synthetase: The Carbonylation of 7,8-Diaminononanoic Acid Proceeds Regiospecifically via the N7-Carbamate

Dethiobiotin synthetase (DTBS) catalyzes the penultimate step in biotin biosynthesis, the formation of the ureido ring of dethiobiotin from (7R,8S)-7,8-diaminononanoic acid (7,8-diaminopelargonic acid, DAPA), CO sub(2), and ATP. Solutions of DAPA at neutral pH readily formed a mixture of the N7- and N8-carbamates in the presence of CO sub(2). However, four lines of evidence together indicated that only the N7-carbamate of DAPA was an intermediate in the reaction catalyzed by DTBS. Addition of diazomethane to mixtures of DAPA and [ super(14)C]CO sub(2) yielded a mixture of the N7- and N8-methyl carbamate esters, consistent with carbamate formation in free solution. In the presence of excess DTBS (over DAPA), the ratio of N7:N8-methyl carbamate esters recovered was roughly doubled, suggesting that the enzyme preferentially bound the N7-DAPA-carbamate. Both N7- and N8-DAPA-carbamates were observed directly by super(1)H and super(13)C NMR in solutions containing DAPA and [ super(13)C]CO sub(2). In the presence of excess DTBS (over DAPA) only one carbamate was observed, showing that carbamate binding to the enzyme was regiospecific. super(13)C NMR of mixtures containing enzyme, [7- super(15)N]DAPA, and [ super(13)C]CO sub(2) showed that the enzyme-bound carbamate was at N7 of DAPA. In addition, pulse-chase experiments showed that the binary complex of DTBS and N7-DAPA-carbamate became kinetically committed upon addition of MgATP. The N7-DAPA-carbamate mimic, 3-(1-aminoethyl)nonanedioic acid, in which the carbamate nitrogen was replaced with a methylene group, cyclized to the corresponding lactam in the presence of DTBS and ATP; ADP and P sub(i) were also formed. The K sub(M) and V sub(max) for this process were comparable to those for the natural substrate, DAPA. By contrast, the N8-DAPA-carbamate mimic, 4-amino-3-methyldecanedioic acid, was a much poorer substrate (V/K less than or equal to 0.1% of that for DAPA), and the compound was only weakly inhibitory. These experiments strongly suggest that DTB is formed predominantly through the N7-carbamate of DAPA. Crystallographic analysis at 1.65 angstrom resolution of several DTBS-DAPA complexes also reveals electron density consistent with the presence of a carbamate on the 7-amino group.