PMID:7669755
| Citation |
Gibson, KJ, Lorimer, GH, Rendina, AR, Taylor, WS, Cohen, G, Gatenby, AA, Payne, WG, Roe, DC, Lockett, BA and Nudelman, A (1995) Dethiobiotin synthetase: the carbonylation of 7,8-diaminonanoic acid proceeds regiospecifically via the N7-carbamate. Biochemistry 34:10976-84 |
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| Abstract |
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.(ABSTRACT TRUNCATED AT 250 WORDS) |
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| Keywords |
Amino Acids, Diamino/chemistry; Amino Acids, Diamino/metabolism; Carbamates/chemistry; Carbamates/metabolism; Carbon-Nitrogen Ligases; Escherichia coli/enzymology; Escherichia coli/genetics; Gas Chromatography-Mass Spectrometry; Kinetics; Ligases/genetics; Ligases/metabolism; Molecular Structure |
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