Probing the penicillin sidechain selectivity of recombinant deacetoxycephalosporin C synthase.
Deacetoxycephalosporin C synthase from Streptomyces clavuligerus catalyses the conversion of the five-membered penicillin ring to the unsaturated six-membered cephem ring of deacetoxycephalosporin C. The effects on enzyme activity of the penicillin substrate sidechain and various cofactors were investigated using a continuous spectrophotometric assay. The conversion of penicillin G to phenylacetyl-7-aminodeacetoxycephalo sporanic acid (G-7-ADCA) was confirmed, and further details of the reaction were elucidated. The conversion of ampicillin to cephalexin was faster than that of acetyl-6-APA to acetyl-7-ADCA kcat = 0.120 +/- 0.001 s(-1) versus 0.035 +/- 0.001 s(-1), but they had similar Km values: 4.86 +/- 0.12 and 3.28 +/- 0.26 mM, respectively. Amoxycillin and penicillin V were also converted at low levels. Conversion was not detected for penicillanate, 6-aminopenicillanate, carbenicillin, temocillin, ticarcillin or benzylpenicilloic acid, suggesting that the enzyme has a relatively strict selectivity for the sidechain of the penicillin substrate.
Dubus
A
A
Centre d'Ingénierie des Protéines, Université de Liège, Institut de Chimie, Belgium. a.dubus@ulg.ac.be
Lloyd
M D
MD
Lee
H J
HJ
Schofield
C J
CJ
Baldwin
J E
JE
Frere
J M
JM
D000658:Amoxicillin / Q000378:metabolism; D000667:Ampicillin / Q000378:metabolism; D001205:Ascorbic Acid / Q000378:metabolism; D002245:Carbon Dioxide / Q000378:metabolism; D002506:Cephalexin / Q000378:metabolism; D002511:Cephalosporins / Q000737:chemistry / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003067:Coenzymes / Q000378:metabolism; D019751:Intramolecular Transferases / Q000235:genetics / Q000378:metabolism*; D007501:Iron / Q000378:metabolism; D007656:Ketoglutaric Acids / Q000378:metabolism; D007700:Kinetics; D010100:Oxygen / Q000378:metabolism; D010400:Penicillin G / Q000378:metabolism; D010404:Penicillin V / Q000378:metabolism; D046915:Penicillin-Binding Proteins; D010406:Penicillins / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D019163:Reducing Agents / Q000378:metabolism; D013053:Spectrophotometry; D013302:Streptomyces / Q000201:enzymology*; D013379:Substrate Specificity; D013816:Thermodynamics
D002511:Cephalosporins; D003067:Coenzymes; D007656:Ketoglutaric Acids; D046915:Penicillin-Binding Proteins; D010406:Penicillins; D011994:Recombinant Proteins; D019163:Reducing Agents; D002245:Carbon Dioxide; C025578:deacetoxycephalosporin C; D000667:Ampicillin; D000658:Amoxicillin; C014320:7-aminodesacetoxycephalosporanic acid; D007501:Iron; D019751:Intramolecular Transferases; C038371:deacetoxycephalosporin C synthetase; D002506:Cephalexin; D001205:Ascorbic Acid; D010400:Penicillin G; D010100:Oxygen; D010404:Penicillin V