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), CO₂, and ATP. Solutions of DAPA at neutral pH readily formed a mixture of the N7- and N8-carbamates in the presence of CO₂. 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]CO₂ 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 ¹H and ¹³C NMR in solutions containing DAPA and [¹³C]CO₂. 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]CO₂ 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 P_i were also formed. The K_m and V_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 ≤ 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 Å 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, 10985-10995].