Comprehensive research of amino acid residues that participate in increasing activity of carbapenemase and structural analysis of mutant enzymes
| Project/Area Number |
17K09018
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Multi-year Fund |
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| Section | 一般 |
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| Research Field |
Laboratory medicine
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| Research Institution | Nara Medical University |
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Principal Investigator |
Yamamoto Keizo 奈良県立医科大学, 医学部, 准教授 (90254490)
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| Co-Investigator(Kenkyū-buntansha) |
矢野 寿一 奈良県立医科大学, 医学部, 教授 (20374944)
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| Project Period (FY) |
2017-04-01 – 2020-03-31
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| Project Status |
Completed (Fiscal Year 2019)
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| Budget Amount *help |
¥4,810,000 (Direct Cost: ¥3,700,000、Indirect Cost: ¥1,110,000)
Fiscal Year 2019: ¥1,040,000 (Direct Cost: ¥800,000、Indirect Cost: ¥240,000)
Fiscal Year 2018: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000)
Fiscal Year 2017: ¥2,600,000 (Direct Cost: ¥2,000,000、Indirect Cost: ¥600,000)
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| Keywords | βーラクタマーゼ / X線結晶構造解析 / 指向性進化法 / カルバペネマーゼ / 基質特異性 / 構造解析 / 感染症 / 人工進化 |
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| Outline of Final Research Achievements |
We performed crystallization and structural analysis of IMP-6 metallo-b-lactamase at a resolution of 0.183 nm. The overall structure of IMP-6 resembled that of IMP-1. Major structural difference was found in the loop region (Glu60-Val66) involved in substrate/inhibitor binding. The binding of meropenem to IMP-6 is stabilized by a hydrophobic interaction between meropenem and the side chain of Trp64. In contrast, our simulation of imipenem binding to the substrate-binding site of IMP-6 showed that steric hindrance between N16 on the R2 side chain of imipenem and NE1 of Trp64 is highly likely.
A Y180N mutant was developed using random mutagenesis by PCR. This mutant enzyme shows higher meropenem-hydrolyzing activity and lower imipenem-hydrolyzing activity compared to those of IMP-6. The amino acid substitution appears to increase flexibility of the loop between the N-terminal and C-terminal domains owing to disruption of pi-stacking interactions.
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| Academic Significance and Societal Importance of the Research Achievements |
本研究の学術的意義は、IMP-6のX線結晶構造解析とシミュレーションの結果とから、IMP-6がイミペネムに対して活性が低い原因が推定されたことと、指向性進化法の手法を用いてメロペネムの選択圧下でIMP-6を進化させると、メロペネムに対する活性がさらに増加することを示したことである。
これらの結果から、メロペネムの過度の使用により、さらに強いカルバペネム耐性菌が出現する可能性を示した。また、イミペネムの側鎖に対し、どのような化学的修飾を加えれば、より効果的な抗菌薬の開発が可能かという方向性を与えた。この2点が、本研究の社会的意義であると考える。
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Report
(4 results)
Research Products
(56 results)
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[Journal Article] Environmental presence and genetic characteristics of carbapenemase-producing Enterobacteriaceae from hospital sewage and river water in the Philippines.2020
Author(s)
Suzuki Y, Nazareno PJ, Nakano R, Mondoy M, Nakano A, Bugayong MP, Bilar J, Perez M 5th, Medina EJ, Saito-Obata M, Saito M, Nakashima K, Oshitani H, Yano H
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Journal Title
Appl Environ Microbiol
Volume: 88
Issue: 2
DOI
Related Report
Peer Reviewed / Open Access / Int'l Joint Research
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[Journal Article] Comparison between IMP carbapenemase-producing Enterobacteriaceae and non-carbapenemase-producing Enterobacteriaceae: a multicentre prospective study of the clinical and molecular epidemiology of carbapenem-resistant Enterobacteriaceae.2020
Author(s)
Hayakawa K, Nakano R, Hase R, Shimatani M, Kato H, Hasumi J, Doi A, Sekiya N, Nei T, Okinaka K, Kasahara K, Kurai H, Nagashima M, Miyoshi-Akiyama T, Kakuta R, Yano H, Ohmagari N.
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Journal Title
J Antimicrob Chemother.
Volume: 75
Issue: 3
Pages: 697-708
DOI
Related Report
Peer Reviewed
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[Journal Article] Comparison of the inoculum size effects in blaIMP-6 and plasmid-mediated quinolone resistance gene-positive Enterobacteriaceae in Japan.2019
Author(s)
Ogawa Y, Nakano R, Kasahara K, Mizuno T, Hirai N, NakanoA, Suzuki Y, Kakuta N, Masui T, Yano H, Mikasa K
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Journal Title
PLoS ONE
Volume: 14
Issue: 11
Pages: e0225210-e0225210
DOI
Related Report
Peer Reviewed / Open Access
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[Journal Article] Suitability of the Carbapenem Inactivation Method (CIM) to Detect IMP Metallo-β-lactamase-Producing Enterobacteriaceae.2017
Author(s)
K Saito, R Nakano, Y Suzuki, A Nakano, Y Ogawa, S Yonekawa, S Endo, F Mizuno, K Kasahara, K Mikasa, M Kaku, H Yano
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Journal Title
Journal of Clinical Microbiology
Volume: 55
Issue: 4
Pages: 1220-1222
DOI
Related Report
Peer Reviewed / Open Access / Acknowledgement Compliant
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[Presentation] Comparison of IMP carbapenemase-producing and non-carbapenemase-producing Enterobacteriaceae: A multicenter prospective study of clinical and molecular epidemiology in Japan.2019
Author(s)
Hayakawa K, Nakano R, Hase R, Shimatani M, Kato H, Hasumi J, Doi A, Sekiya N, Nei T, Okinaka K, Kasahara K, Kurai H, Nagashima M, Miyoshi-Akiyama T, Kakuta R, Yano H, Ohmagari N
Organizer
IDWeek
Related Report
Int'l Joint Research
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[Presentation] Diversity of CTX-M β-lactamase genotypes among IMP-6 β-lactamase-producing Escherichia coli in Japan2018
Author(s)
Mizuno T, Nakano R, Ogawa M, Suzuki Y, Nakano A, Ando S, Tanouchi A, Kakuta N, Masui T, Saito K, Kasahara K, Yano H
Organizer
28th European Congress of Clinical Microbiology and Infectious Diseases
Related Report
Int'l Joint Research
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[Presentation] Accuracy of carbapenem inactivation method (CIM) for detecting carbapenemase-producing Gram-negative bacteria corresponding to bacterial species and enzymes.2017
Author(s)
Saito K, Nakano R, Suzuki Y, Nakano A, Yonekawa S, Endo S, Mizuno F, Kasahara K, Mikasa K, Kaku M, Yano H
Organizer
27th European Congress of Clinical Microbiology and Infectious Diseases
Related Report
Int'l Joint Research
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