Role of Asp104 in the SHV beta-lactamase.
Among the TEM-type extended-spectrum beta-lactamases (ESBLs), an amino acid change at Ambler position 104 (Glu to Lys) results in increased resistance to ceftazidime and cefotaxime when found with other substitutions (e.g., Gly238Ser and Arg164Ser). To examine the role of Asp104 in SHV beta-lactamases, site saturation mutagenesis was performed. Our goal was to investigate the properties of amino acid residues at this position that affect resistance to penicillins and oxyimino-cephalosporins. Unexpectedly, 58% of amino acid variants at position 104 in SHV expressed in Escherichia coli DH10B resulted in beta-lactamases with lowered resistance to ampicillin. In contrast, increased resistance to cefotaxime was demonstrated only for the Asp104Arg and Asp104Lys beta-lactamases. When all 19 substitutions were introduced into the SHV-2 (Gly238Ser) ESBL, the most significant increases in cefotaxime and ceftazidime resistance were noted for both the doubly substituted Asp104Lys Gly238Ser and the doubly substituted Asp104Arg Gly238Ser beta-lactamases. Correspondingly, the overall catalytic efficiency (kcat/Km) of hydrolysis for cefotaxime was increased from 0.60 +/- 0.07 microM(-1) s(-1) (mean +/- standard deviation) for Gly238Ser to 1.70 +/- 0.01 microM(-1) s(-1) for the Asp104Lys and Gly238Ser beta-lactamase (threefold increase). We also showed that (i) k3 was the rate-limiting step for the hydrolysis of cefotaxime by Asp104Lys, (ii) the Km for cefotaxime of the doubly substituted Asp104Lys Gly238Ser variant approached that of the Gly238Ser beta-lactamase as pH increased, and (iii) Lys at position 104 functions in an energetically additive manner with the Gly238Ser substitution to enhance catalysis of cephalothin. Based on this analysis, we propose that the amino acid at Ambler position 104 in SHV-1 beta-lactamase plays a major role in substrate binding and recognition of oxyimino-cephalosporins and influences the interactions of Tyr105 with penicillins.
Bethel
Christopher R
CR
Louis Stokes Cleveland Veterans Affairs Medical Center, 10701 East Blvd., Cleveland, OH 44106, USA.
Hujer
Andrea M
AM
Hujer
Kristine M
KM
Thomson
Jodi M
JM
Ruszczycky
Mark W
MW
Anderson
Vernon E
VE
Pusztai-Carey
Marianne
M
Taracila
Magdalena
M
Helfand
Marion S
MS
Bonomo
Robert A
RA
D019943:Amino Acid Substitution; D001216:Asparagine / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002384:Catalysis; D024901:Drug Resistance, Multiple, Bacterial; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification
D001216:Asparagine; C054882:beta-lactamase PIT-2; D001618:beta-Lactamases