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Antimicrobial Agents and Chemotherapy, December 2006, p. 4124-4131, Vol. 50, No. 12
0066-4804/06/$08.00+0     doi:10.1128/AAC.00848-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Role of Asp104 in the SHV ß-Lactamase{triangledown}

Christopher R. Bethel,1 Andrea M. Hujer,1 Kristine M. Hujer,1 Jodi M. Thomson,2 Mark W. Ruszczycky,3 Vernon E. Anderson,3 Marianne Pusztai-Carey,3 Magdalena Taracila,1 Marion S. Helfand,1,3 and Robert A. Bonomo1,2*

Louis Stokes Cleveland Veterans Affairs Medical Center, Cleveland, Ohio,1 Departments of Pharmacology,2 Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, Ohio3

Received 12 July 2006/ Returned for modification 9 August 2006/ Accepted 7 September 2006

Among the TEM-type extended-spectrum ß-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 ß-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 ß-lactamases with lowered resistance to ampicillin. In contrast, increased resistance to cefotaxime was demonstrated only for the Asp104Arg and Asp104Lys ß-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 ß-lactamases. Correspondingly, the overall catalytic efficiency (kcat/Km) of hydrolysis for cefotaxime was increased from 0.60 ± 0.07 µM–1 s–1 (mean ± standard deviation) for Gly238Ser to 1.70 ± 0.01 µM–1 s–1 for the Asp104Lys and Gly238Ser ß-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 ß-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 ß-lactamase plays a major role in substrate binding and recognition of oxyimino-cephalosporins and influences the interactions of Tyr105 with penicillins.

* Corresponding author. Mailing address: Infectious Diseases Section, Louis Stokes Cleveland Veterans Affairs Medical Center, 10701 East Blvd., Cleveland, OH 44106. Phone: (216) 791-3800, ext. 4399. Fax: (216) 231-3482. E-mail: robert.bonomo{at}med.va.gov.

{triangledown} Published ahead of print on 18 September 2006.

Antimicrobial Agents and Chemotherapy, December 2006, p. 4124-4131, Vol. 50, No. 12
0066-4804/06/$08.00+0     doi:10.1128/AAC.00848-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Pattanaik, P., Bethel, C. R., Hujer, A. M., Hujer, K. M., Distler, A. M., Taracila, M., Anderson, V. E., Fritsche, T. R., Jones, R. N., Pagadala, S. R. R., van den Akker, F., Buynak, J. D., Bonomo, R. A. (2009). Strategic Design of an Effective {beta}-Lactamase Inhibitor: LN-1-255, A 6-ALKYLIDENE-2'-SUBSTITUTED PENICILLIN SULFONE. J. Biol. Chem. 284: 945-953 [Abstract] [Full Text]  
  • Thomson, J. M., Prati, F., Bethel, C. R., Bonomo, R. A. (2007). Use of Novel Boronic Acid Transition State Inhibitors To Probe Substrate Affinity in SHV-Type Extended-Spectrum {beta}-Lactamases. Antimicrob. Agents Chemother. 51: 1577-1579 [Abstract] [Full Text]  


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