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Antimicrobial Agents and Chemotherapy, October 2007, p. 3778-3779, Vol. 51, No. 10
0066-4804/07/$08.00+0     doi:10.1128/AAC.00633-07

LETTER TO THE EDITOR

Exact Location of the Region Responsible for the Extended Substrate Spectrum in Class C ß-Lactamases

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In a recently published article (9), Wachino et al. reported that a single amino acid substitution (I292S) responsible for the expansion of the hydrolyzing activity against several extended-spectrum cephalosporins (ceftazidime or cefepime) is near the H-10 helix region of CMY-19. However, other reports (2, 7, 8) have concluded that the region responsible for the extended substrate spectrum in class C ß-lactamases is in (not near) the H-10 helix.

Wachino and colleagues designated the amino acid sequence from 279 to 287 as the H-10 helix (Fig. 1), which may be based on the crystal structure of AmpC K-12 (PDB code 2BLS), although they did not state the fact. However, one previous report described residues 279 to 294 as the H-10 helix, according to the crystal structure of GC1 (3). Our recent report (5) showed that the sequence from 289 to 294 was the H-10 (10) helix (Fig. 1), based on superposed crystal structures among CMY-10 (5), P99, and GC1 ß-lactamases. There are different positions of the H-10 helix that is related to the extended substrate spectrum in class C ß-lactamases. Therefore, we propose that the exact region responsible for the extended substrate spectrum is the R2 loop (residues 289 to 307) (Fig. 1) described in our recent report (5). There are three important reasons why the exact region is the R2 loop in the R2 active site, referring to the region that accommodates the R2 side chain at C3 of the ß-lactam nucleus in extended-spectrum cephalosporins.

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FIG. 1. A sequence alignment of amino acid residues near the H-9 (9) and H-10 helix (10) of class C ß-lactamases with extended substrate spectrum. Alignment among CMY-10, P99, and GC1 ß-lactamases whose structures are available is performed based on their superposed structures. The top of the sequence alignment indicates secondary structure annotation of CMY-10 (5). A partial amino acid sequence alignment of CMY-10 (Enterobacter aerogenes K9911729; GenBank accession no. AF357598; PDB code 1ZKJ), GC1 (Enterobacter cloacae GC1; GenBank accession no. D44479; PDB code 1GCE), P99 (E. cloacae P99; GenBank accession no. X07274; PDB code 2BLT), CMY-19 (Klebsiella pneumoniae HKY466; GenBank accession no. AB194410), CMY-9 (K. pneumoniae HKY209; GenBank accession no. AB061794), CMY-11 (Escherichia coli K983802; GenBank accession no. AF357600), FOX-1 (K. pneumoniae BA32; GenBank accession no. X77455), Ear1 (E. cloacae Ear1; GenBank accession no. AJ544161), Ear2 (E. cloacae Ear2; GenBank accession no. AJ544162), CHE (E. cloacae CHE; GenBank accession no. AJ278994), K-12 (E. coli K-12; GenBank accession no. U00096), HKY28 (E. coli HKY28; GenBank accession no. AB108683), S3 (Serratia marcescens S3; GenBank accession no. AF327324), and HD (S. marcescens HD; GenBank accession no. AY336102) is shown. H-10 helix regions described by Wachino et al. (9) and Kim et al. (5) are light gray and dark gray, respectively. The R2 loop (5) of residues 289 to 307 is doubly underlined.

First, the R2 loop includes all regions responsible for the extended substrate spectrum in all reported class C extended-spectrum ß-lactamases except HKY28 (4): (i) six-amino-acid deletion (residues 289 to 294) of CHE (2); (ii) the single amino acid substitution (L296H) of AmpC KL (7); (iii) four-amino-acid deletion (residues 293 to 296) of HD (8); (iv) three-amino-acid deletion (residues 303 to 305) of CMY-10 (5); (v) the single amino acid substitution (I292S) of CMY-19 (9); and (vi) the L293P substitution of Ear2 (1).

Second, mutations in the R2 loop can change the architecture of the active site in class C extended-spectrum ß-lactamases, thereby affecting their hydrolyzing 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 ß11 (Fig. 1) induces a 2.5-Å shift of 9 and 10 relative to the adjacent helix 11 in CMY-10, compared with both P99 and GC1 ß-lactamases, opening the gap between 9-10 and 11 (5). Therefore, the bulky R2 side chain of extended-spectrum cephalosporins could fit snugly into the significantly widened R2 active site in this way (6).

Third, CMY-19 showed 97% sequence identity to CMY-10. But the sequence identity between CMY-19 and AmpC K-12 was 40%. Therefore, it is reasonable for the region responsible for the extended substrate spectrum in CMY-19 to be designated based on the crystal structure of CMY-10 (not AmpC K-12).

 
We acknowledge the financial support of the National Institute of Health (NIH) of KCDC in Republic of Korea, beamline 6B and 6C of PLS, supported by MOST and POSCO, the 21C Frontier Functional Proteomics Center, the Driving Force Project for the Next Generation of Gyeonggi Provincial Government in the Republic of Korea, the Korea Research Foundation (KRF-2006-331-E00455), and the Second-Phase of Brain Korea 21 Project.

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1. Barnaud, G., Y. Benzerara, J. Gravisse, L. Raskine, M. J. Sanson-Le Pors, G. Labia, and G. Arlet. 2004. Selection during cefepime treatment of a new cephalosporinase variant with extended-spectrum resistance to cefepime in an Enterobacter aerogenes clinical isolate. Antimicrob. Agents Chemother. 48:1040-1042.[Abstract/Free Full Text]
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2. Barnaud, G., R. Labia, L. Raskine, M. J. Sanson-Le Pors, A. Philippon, and G. Arlet. 2001. Extension of resistance to cefepime and cefpirome associated to a six amino acid deletion in the H-10 helix of the cephalosporinase of an Enterobacter cloacae clinical isolate. FEMS. Microbiol. Lett. 195:185-190.[CrossRef][Medline]
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3. Crichlow, G. V., A. P. Kuzin, M. Nukaga, K. Mayama, T. Sawai, and J. R. Knox. 1999. Structure of the extended-spectrum class C ß-lactamase of Enterobacter cloacae GC1, a natural mutant with a tandem tripeptide insertion. Biochemistry 38:10256-10261.[CrossRef][Medline]
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4. Doi, Y., J. Wachino, M. Ishiguro, H. Kurokawa, K. Yamane, N. Shibata, K. Shibayama, K. Yokoyama, H. Kato, T. Yagi, and Y. Arakawa. 2004. Inhibitor-sensitive AmpC ß-lactamase variant produced by an Escherichia coli clinical isolate resistant to oxyiminocephalosporins and cephamycins. Antimicrob. Agents. Chemother. 48:2652-2658.[Abstract/Free Full Text]
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5. Kim, J. Y., H. I. Jung, Y. J. An, J. H. Lee, S. J. Kim, S. H. Jeong, K. J. Lee, P. G. Suh, H. S. Lee, S. H. Lee, and S. S. Cha. 2006. Structural basis for the extended substrate spectrum of CMY-10, a plasmid-encoded class C ß-lactamase. Mol. Microbiol. 60:907-916.[CrossRef][Medline]
6
6. Lee, S. H., S. H. Jeong, and S. S. Cha. 2005. Minimising antibiotic resistance. Lancet Infect. Dis. 5:668-670.[CrossRef][Medline]
7
7. Mammeri, H., H. Nazic, T. Naas, L. Poirel, S. Leotard, and P. Nordmann. 2004. AmpC beta-lactamase in an Escherichia coli clinical isolate confers resistance to expanded-spectrum cephalosporins. Antimicrob. Agents Chemother. 48:4050-4053.[Abstract/Free Full Text]
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8. Mammeri, H., L. Poirel, P. Bemer, H. Drugeon, and P. Nordmann. 2004. Resistance to cefepime and cefpirome due to a 4-amino-acid deletion in the chromosome-encoded AmpC ß-lactamase of a Serratia marcescens clinical isolate. Antimicrob. Agents. Chemother. 48:716-720.[Abstract/Free Full Text]
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9. Wachino, J., H. Kurokawa, S. Suzuki, K. Yamane, N. Shibata, K. Kimura, Y. Ike, and Y. Arakawa. 2006. Horizontal transfer of bla_CMY-bearing plasmids among clinical Escherichia coli and Klebsiella pneumoniae isolates and emergence of cefepime-hydrolyzing CMY-19. Antimicrob. Agents. Chemother. 50:534-541.[Abstract/Free Full Text]

						Sang Hee Lee* Jung Hun Lee Myong Jin Heo Department of Biological Sciences Myongji University San 38-2 Namdong, Yongin Gyeonggido 449-728, Republic of Korea Il Kwon Bae Seok Hoon Jeong Department of Laboratory Medicine College of Medicine Kosin University Busan 602-702, Republic of Korea Sun-Shin Cha Structural Biology Laboratory Marine Biotechnology Center Korea Ocean Research & Development Institute Ansan, Republic of Korea
						* Phone: 82 31 330 6195, Fax: 82 31 335 8249, E-mail: sangheelee{at}mju.ac.kr

Authors’ Reply

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We would like to express our sincere thanks to Dr. Lee and his colleagues for their perusal of our paper describing the CMY-19 class C ß-lactamase (2). In that article, we first elucidated that a single amino acid substitution from I to S at the amino acid position 292 found in this enzyme is responsible for its extended substrate specificity against cefepime, as well as other oxyiminocephalosporins. As for the location of the H-10 helix in CMY-19, we simply referred to the crystal structure of AmpC of Escherichia coli K-12, because no tertiary structure of CMY enzymes was available when we submitted our manuscript. A fine crystallographic structure of the CMY-10 cephamycinase was first reported in May 2006 (1), 3 months later than our publication about CMY-19 (2). Dr. Kim et al. explicated the exact 10 helix domain in CMY-10, and the 292S residue of CMY-19 is certainly located in the 10 helix. As a matter of importance, deletions or substitutions of amino acid residues in the -helix region consisting of the 9 and 10 domains of CMY enzymes are crucial for expansion of substrate specificity among CMY-type enzymes. The region containing both 9 and 10 of CMY-10 corresponds to the previously described H-10 helix of K-12 AmpC, which is considerably different from CMY enzymes. At any rate, our concern is why the single amino acid substitution I292S affects the substrate specificity of CMY enzymes for cefepime, and we hope Dr. Lee and his colleagues will further elucidate the fine molecular structure of CMY-11, which has an I292S substitution very similar to that of CMY-19.

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1. Kim, J. Y., H. I. Jung, Y. J. An, J. H. Lee, S. J. Kim, S. H. Jeong, K. J. Lee, P. G. Suh, H. S. Lee, S. H. Lee, and S. S. Cha. 2006. Structural basis for the extended substrate spectrum of CMY-10, a plasmid-encoded class C ß-lactamase. Mol. Microbiol. 60:907-916.[CrossRef][Medline]
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2. Wachino, J., H. Kurokawa, S. Suzuki, K. Yamane, N. Shibata, K. Kimura, Y. Ike, and Y. Arakawa. 2006. Horizontal transfer of bla_CMY-bearing plasmids among clinical Escherichia coli and Klebsiella pneumoniae isolates and emergence of cefepime-hydrolyzing CMY-19. Antimicrob Agents Chemother. 50:534-541.[Abstract/Free Full Text]

						Jun-ichi Wachino Yoshichika Arakawa Department of Bacteria Pathogenesis and Infection Control National Institute of Infectious Diseases 4-7-1 Gakuen, Musashi-Murayama Tokyo 208-0011, Japan

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Antimicrobial Agents and Chemotherapy, October 2007, p. 3778-3779, Vol. 51, No. 10
0066-4804/07/$08.00+0     doi:10.1128/AAC.00633-07

This article has been cited by other articles:

• Jacoby, G. A. (2009). AmpC {beta}-Lactamases. Clin. Microbiol. Rev. 22: 161-182 [Abstract] [Full Text]  
• Sohn, S. G., Lee, J. J., Sohn, E. S., Kang, L.-W., Lee, S. H. (2008). Comment on: Extension of the hydrolysis spectrum of AmpC {beta}-lactamase of Escherichia coli due to amino acid insertion in the H-10 helix. J Antimicrob Chemother 61: 965-966 [Full Text]  

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