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), C02, and ATP. Solutions of DAPA at neutral pH readily formed a mixture of the N7- and N8-carbamates in the presence of C02. However, four lines of evidence together indicated that only the N7-carbamate of DAPA was an intermediate in the reaction catalyzed by DTBS. (1) Addition of diazomethane to mixtures of DAPA and (14C)C02 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. (2) Both N7- and N8-DAPA-carbamates were observed directly by lH and 13C NMR in solutions containing DAPA and (13C)C02. In the presence of excess DTBS (over DAPA) only one carbamate was observed, showing that carbamate binding to the enzyme was regiospecific. 13C NMR of mixtures containing enzyme, (7-15N)DAPA, and (13C)C02 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. (3) 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 Pi were also formed. The KM and V,,, for this process were comparable to those for the natural substrate, DAPA. By contrast, the N8-DAPA-carbamate mimic, 4-amho-3- methyldecanedioic acid, was a much poorer substrate (VIK I 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. (4) Crystallographic analysis at 1.65 8, resolution of several DTBS-DAPA complexes also reveals electron density consistent with the presence of a carbamate on the 7-amino group (Huang, W., Jia, J., Gibson, K. J., Taylor, W. S., Rendina, A. R., Schneider, G., & Lindqvist, Y. (1995) Biochemistry 34, 10985 - 109951.