JAC Advance Access originally published online on January 27, 2008
Journal of Antimicrobial Chemotherapy 2008 61(4):965-966; doi:10.1093/jac/dkn023
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Letters to the Editor |
Comment on: Extension of the hydrolysis spectrum of AmpC β-lactamase of Escherichia coli due to amino acid insertion in the H-10 helix
1 Department of Biological Sciences, Myongji University, San 38-2 Namdong, Yongin, Gyeonggido 449-728, Republic of Korea 2 Division of Antimicrobial Resistance, Center for Infectious Disease Research, National Institute of Health, 194 Tongillo, Seoul 122-701, Republic of Korea 3 Structural Biology Laboratory, Department of Advanced Technology Fusion, Konkuk University, 1 Hwayangdong, Seoul 143-701, Republic of Korea
* Corresponding author. Tel: +82-31-330-6195; Fax: +82-31-335-8249; E-mail: sangheelee{at}mju.ac.kr
Keywords: R2 loop , extended substrate spectrum , class C β-lactamase
Sir,
Mammeri et al.1 recently reported that the 2 amino acid insertion, responsible for the expansion of the hydrolysing activity against several extended-spectrum (ES) cephalosporins (ceftazidime, cefotaxime or cefepime), was within the H-10 helix region of chromosomal AmpC BER (BER). However, another report2 concluded that the region responsible for the extended substrate spectrum in class C β-lactamases was near (but not within) the H-10 helix of the plasmid-encoded CMY-19.
Why is the region responsible for an extended substrate spectrum located in different positions of the H-10 helix region of different class C β-lactamases? The reason is that there are different designations of where the H-10 helix resides, as follows: (i) the amino acid sequence from 279 to 287 as the H-10 helix in the case of CMY-19;2 (ii) the residues 279–294 as the H-10 helix in the case of the chromosomal AmpC GC1;3 and (iii) in another article reported by Mammeri and Nordmann,4 the residues 280–293 as the H-10 helix in the case of the chromosomal ES AmpC β-lactamases. Our recent report3 showed that the sequence from 289 to 294 is the actual location of the H-10 (
10) helix, based on superposed crystal structures among the plasmid-encoded CMY-10 and AmpC P99 (P99) β-lactamases. Therefore, we suggested that the exact region responsible for the extended substrate spectrum is not the H-10 helix.3 The R2 loop does include H-10 (Figure 1), and thus the results of Mammeri et al.1 are consistent with our recent report.3 The R2 loop defines the edge of the R2 active site referring to the region that accommodates the R2 side-chain at C3 of the β-lactam nucleus in ES cephalosporins. Our sequence alignment (Figure 1) of four class C ES β-lactamases shows that the R2 loop includes all regions responsible for the extended substrate spectrum in four class C ES β-lactamases, compared with P99 (a class C non-ES β-lactamase): (i) three amino acid deletions (residues 303–305) of CMY-10; (ii) four amino acid deletions (residues 293–296) of chromosomal AmpC HD (HD); (iii) two amino acid insertions between residues 293 and 294 of BER; and (iv) the single amino acid substitution (A292S) and three amino acid deletions (residues 303–305) of CMY-19. Mutations in the R2 loop can change the architecture of the active site in class C ES β-lactamases, thereby affecting their hydrolysing activity. Owing to the deletion in CMY-10, for example, the R2 loop in the R2 active site displays noticeable structural alterations: the shortened path of the connection R2 loop between 10 and β11 induces the significant widening of the R2 active site in CMY-10 when compared with the P99 β-lactamase.3,5 In addition, the bulky R2 side-chain of ES cephalosporins could fit snugly into the significant widening of the R2 active site in this way. Therefore, the facts described above suggest that the region responsible for the extended substrate spectrum is the R2 loop (residues 289–310 according to the amino acid numbering system of P99; Figure 1).
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Another issue that we wish to highlight from our reading of the work of Mammeri et al.1 is a commonly made oversimplification that a decrease in Km indicates an increased affinity of a substrate for an enzyme. Because an increased k2/k3 ratio (not only decreased Km value) also yields an increased affinity,6 the Km value is a complex constant that does not simply reflect the binding affinity, especially when the deacylation step (related to k2 and k3) is rate-limiting.
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The work carried out on CMY-10 was funded in part by grants from the National Institute of Health (NIH) of KCDC in Republic of Korea, BioGreen 21 Program (Z0070501034003) of Rural Development Administration in Republic of Korea and the Driving Force Project for the Next Generation of Gyeonggi Provincial Government in Republic of Korea.
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None to declare.
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1 Mammeri H, Poirel L, Nordmann P. Extension of the hydrolysis spectrum of AmpC β-lactamase of Escherichia coli due to amino acid insertion in the H-10 helix. J Antimicrob Chemother (2007) 60:490–4.
2
Wachino J-I, Kurokawa H, Suzuki S, et al. Horizontal transfer of blaCMY-bearing plasmids among clinical Escherichia coli and Klebsiella pneumoniae isolates and emergence of cefepime-hydrolyzing CMY-19. Antimicrob Agents Chemother (2006) 50:534–41.
3
Lee SH, Lee JH, Heo MJ, et al. Exact location of the region responsible for the extended substrate spectrum in class C β-lactamases. Antimicrob Agents Chemother (2007) 51:3778–9.
4 Mammeri H, Nordmann P. Extended-spectrum cephalosporinases in Enterobacteriaceae. Anti-Infect Agents Med Chem (2007) 6:71–82.
5 Kim JY, Jung HI, An YJ, et al. Structural basis for the extended substrate spectrum of CMY-10, a plasmid-encoded class C β-lactamase. Mol Microbiol (2006) 60:907–16.[CrossRef][Web of Science][Medline]
6 Galleni M, Frere J-M. Kinetics of β-lactamases and penicillin-binging proteins. In: Enzyme-Mediated Resistance to Antibiotics: Mechanisms, Dissemination, and Prospects for Inhibition—Bonomo RA, Tolmasky ME, eds. (2007) Washington, DC: ASM Press. 195–213.
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