2019 Fiscal Year Final Research Report

Comprehensive research of amino acid residues that participate in increasing activity of carbapenemase and structural analysis of mutant enzymes

Research Project

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Project/Area Number	17K09018
Research Category	Grant-in-Aid for Scientific Research (C)
Allocation Type	Multi-year Fund
Section	一般
Research Field	Laboratory medicine
Research Institution	Nara Medical University
Principal Investigator	Yamamoto Keizo 奈良県立医科大学, 医学部, 准教授 (90254490)
Co-Investigator(Kenkyū-buntansha)	矢野寿一奈良県立医科大学, 医学部, 教授 (20374944)
Project Period (FY)	2017-04-01 – 2020-03-31
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
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.
Free Research Field	タンパク質工学
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。