title: Dethiobiotin synthetase: the carbonylation of 7,8-diaminonanoic acid proceeds regiospecifically via the N7-carbamate.
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)
authors.affiliation: Dupont Central Research and Development, Experimental Station, Wilmington, Delaware 19880-0407, USA.
authors.forename: K J
authors.initials: KJ
authors.lastname: Gibson
authors.forename: G H
authors.initials: GH
authors.lastname: Lorimer
authors.forename: A R
authors.initials: AR
authors.lastname: Rendina
authors.forename: W S
authors.initials: WS
authors.lastname: Taylor
authors.forename: G
authors.initials: G
authors.lastname: Cohen
authors.forename: A A
authors.initials: AA
authors.lastname: Gatenby
authors.forename: W G
authors.initials: WG
authors.lastname: Payne
authors.forename: D C
authors.initials: DC
authors.lastname: Roe
authors.forename: B A
authors.initials: BA
authors.lastname: Lockett
authors.forename: A
authors.initials: A
authors.lastname: Nudelman
mesh_terms: D000599:Amino Acids, Diamino / Q000737:chemistry / Q000378:metabolism*; D002219:Carbamates / Q000737:chemistry / Q000378:metabolism; D019731:Carbon-Nitrogen Ligases; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D008401:Gas Chromatography-Mass Spectrometry; D007700:Kinetics; D008025:Ligases / Q000235:genetics / Q000378:metabolism*; D015394:Molecular Structure
chemical_list: D000599:Amino Acids, Diamino; D002219:Carbamates; C004720:7,8-diaminopelargonic acid; D008025:Ligases; D019731:Carbon-Nitrogen Ligases; C020024:dethiobiotin synthetase