A novel esterase from Burkholderia gladioli which shows high deacetylation activity on cephalosporins is related to beta-lactamases and DD-peptidases.
The gene (estB) encoding for a novel esterase (EstB) from Burkholderia gladioli (formerly Pseudomonas marginata) NCPPB 1891 was cloned in Escherichia coli. Sequence analysis showed an open reading frame encoding a polypeptide of 392 amino acid residues, with a molecular mass of about 42 kDa. Comparison of the amino acid sequence with those of other homologous enzymes indicated homologies to beta-lactamases, penicillin binding proteins and DD-peptidases. The serine residue (Ser(75)) which is located within a present class A beta-lactamase motif ([F,Y]-X-[L,I,V,M,F,Y]-X-S-[T,V]-X-K-X-X-X-X-[A,G,L]-X-X-[L,C]) was identified by site-directed mutagenesis to represent the active nucleophile. A second serine residue (Ser(149)) which is located within a G-x-S-x-G motif which is typically found in esterases and lipases was demonstrated not to play a significant role in enzyme function. The estB gene was overexpressed in E. coli using a tac promoter-based expression system. Investigation of EstB protein with respect to the ability to hydrolyse beta-lactam substrates clearly demonstrated that this protein has no beta-lactamase activity. The recombinant enzyme is active on triglycerides and on nitrophenyl esters with acyl chain lengths up to C6. The preference for short chain length substrates indicated that EstB is a typical carboxylesterase. As a special feature EstB esterase was found to have high deacetylation activity on cephalosporin derivatives.
Petersen
E I
EI
Institut für Biotechnologie, AG Genetik, SFB Biokatalyse, Technische Universität Graz, Petersgasse 12, A-8010 Graz, Austria.
Valinger
G
G
Sölkner
B
B
Stubenrauch
G
G
Schwab
H
H
D000107:Acetylation; D000595:Amino Acid Sequence; D001483:Base Sequence; D019117:Burkholderia / Q000201:enzymology*; D002268:Carboxypeptidases / Q000378:metabolism*; D002511:Cephalosporins / Q000378:metabolism*; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016366:Open Reading Frames; D011401:Promoter Regions, Genetic; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D046929:Serine-Type D-Ala-D-Ala Carboxypeptidase; D001618:beta-Lactamases / Q000378:metabolism*
D002511:Cephalosporins; D017931:DNA Primers; D012694:Serine; D002268:Carboxypeptidases; D046929:Serine-Type D-Ala-D-Ala Carboxypeptidase; D012697:Serine Endopeptidases; C049262:esterase B; D001618:beta-Lactamases