Probing the Role of Asp-120(81) of Metallo-β-lactamase (IMP-1) by Site-directed Mutagenesis, Kinetic Studies, and X-ray Crystallography

Yamaguchi, Yoshihiro; Kuroki, Takahiro; Yasuzawa, Hisami; Higashi, Toshihiro; Jin, Wei; Kawanami, Akiko; Yamagata, Yuriko; Arakawa, Yoshichika; Goto, Masafumi; Kurosaki, Hiromasa

doi:10.1074/jbc.m414314200

Cited by 61 publications

(87 citation statements)

References 48 publications

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“…Several mutants of Asp120 in the class B3 MBL enzyme L1 from Stenotrophomonas maltophilia showed a 10-to 1,000-fold decrease in k cat compared to wild-type L1, and thus, Asp120 was confirmed to play a significant role in the hydrolysis reaction (22,23). This has also been shown for IMP-1 (24), CphA (25), and BcII (26). To study the role and importance of Asp120 in VIM-7 for zinc binding, thermostability, and catalysis, a D120A mutant was created and analyzed.…”

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confidence: 50%

His224 Alters the R2 Drug Binding Site and Phe218 Influences the Catalytic Efficiency of the Metallo-β-Lactamase VIM-7

Leiros

Skagseth

Edvardsen

et al. 2014

Antimicrob Agents Chemother

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b Metallo-␤-lactamases (MBLs) are the causative mechanism for resistance to ␤-lactams, including carbapenems, in many Gramnegative pathogenic bacteria. One important family of MBLs is the Verona integron-encoded MBLs (VIM). In this study, the importance of residues Asp120, Phe218, and His224 in the most divergent VIM variant, VIM-7, was investigated to better understand the roles of these residues in VIM enzymes through mutations, enzyme kinetics, crystal structures, thermostability, and docking experiments. The tVIM-7-D120A mutant with a tobacco etch virus (TEV) cleavage site was enzymatically inactive, and its structure showed the presence of only the Zn1 ion. The mutant was less thermostable, with a melting temperature (T m ) of 48.5°C, compared to 55.3°C for the wild-type tVIM-7. In the F218Y mutant, a hydrogen bonding cluster was established involving residues Asn70, Asp84, and Arg121. The tVIM-7-F218Y mutant had enhanced activity compared to wild-type tVIM-7, and a slightly higher T m (57.1°C) was observed, most likely due to the hydrogen bonding cluster. Furthermore, the introduction of two additional hydrogen bonds adjacent to the active site in the tVIM-7-H224Y mutant gave a higher thermostability (T m , 62.9°C) and increased enzymatic activity compared to those of the wild-type tVIM-7. Docking of ceftazidime in to the active site of tVIM-7, tVIM-7-H224Y, and VIM-7-F218Y revealed that the side-chain conformations of residue 224 and Arg228 in the L3 loop and Tyr67 in the L1 loop all influence possible substrate binding conformations. In conclusion, the residue composition of the L3 loop, as shown with the single H224Y mutation, is important for activity particularly toward the positively charged cephalosporins like cefepime and ceftazidime.

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His224 Alters the R2 Drug Binding Site and Phe218 Influences the Catalytic Efficiency of the Metallo-β-Lactamase VIM-7

Leiros

Skagseth

Edvardsen

et al. 2014

Antimicrob Agents Chemother

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“…The value of the stereochemical parameter (37), which could discriminate between a square pyramid ( ϭ 0) and trigonal bipyramid ( ϭ 1), is 0.37 { ϭ (34). It must be pointed out that one of the carboxylate oxygen atoms of D120[76] is also involved in hydrogen bonding to the bridging hydroxide oxygen atom (Wat1), suggesting that D120[76] may be particularly important not only in orientating an OH Ϫ acting as a nucleophile to the carbonyl carbon on the ␤-lactam ring but also in positioning the Zn2 and H263 [215], as seen also in the subclass B1 and B3 MBLs (38)(39)(40). These results suggested that SMB-1 has the ability to keep the coordination geometry closer to the transition state through distortion of the regular geometry.…”

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confidence: 99%

Structural Insights into the Subclass B3 Metallo-β-Lactamase SMB-1 and the Mode of Inhibition by the Common Metallo-β-Lactamase Inhibitor Mercaptoacetate

Wachino

Yamaguchi

Mori

et al. 2013

Antimicrob Agents Chemother

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A novel subclass B3 metallo-␤-lactamase (MBL), SMB-1, recently identified from a Serratia marcescens clinical isolate, showed a higher hydrolytic activity against a wide range of ␤-lactams than did the other subclass B3 MBLs, i.e., BJP-1 and FEZ-1, from environmental bacteria. To identify the mechanism underlying the differences in substrate specificity among the subclass B3 MBLs, we determined the structure of SMB-1, using 1.6-Å diffraction data. Consequently, we found that SMB-1 reserves a space in the active site to accommodate ␤-lactam, even with a bulky R1 side chain, due to a loss of amino acid residues corresponding to F31 and L226 of BJP-1, which protrude into the active site to prevent ␤-lactam from binding. The protein also possesses a unique amino acid residue, Q157, which probably plays a role in recognition of ␤-lactams via the hydrogen bond interaction, which is missing in BJP-1 and FEZ-1, whose K m values for ␤-lactams are particularly high. In addition, we determined the mercaptoacetate (MCR)-complexed SMB-1 structure and revealed the mode of its inhibition by MCR: the thiolate group bridges to two zinc ions (Zn1 and Zn2). One of the carboxylate oxygen atoms of MCR makes contact with Zn2 and Ser221, and the other makes contact with T223 and a water molecule. Our results demonstrate the possibility that MCR could be a potent inhibitor for subclass B3 MBLs and that the screening technique using MCR as an inhibitor would be effective for detecting subclass B3 MBL producers.

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“…9-14 Bacteroides fragilis (CcrA), 15-17 Elizabethkingia meningoseptica (BlaB), 18,19 as well as the transferable VIM, IMP, SPM and GIM-type enzymes. [20][21][22][23][24][25][26] includes the CphA and imiS lactamases from Aeromonas species. 27-29 Subclass B3, along with the extensively characterized enzyme L1 from Stenotrophomonas maltophilia ,30-33 includes enzymes from environmental bacteria, such as CAU-1 from Caulobacter crescentus 34 and THIN-B from Janthinobacterium lividum , 35 and from opportunistic pathogens like FEZ-1 from Legionella gormanii ,36-38 and GOB from E. meningoseptica.…”

Section: Introductionmentioning

confidence: 99%

Role of Zinc Content on the Catalytic Efficiency of B1 Metallo β-Lactamases

et al. 2007

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Metallo β-lactamases (MβL's) are enzymes naturally evolved by bacterial strains under the evolutionary pressure of β-lactam antibiotic clinical use. They have a broad substrate spectrum and are resistant to all the clinically useful inhibitors, representing a potential risk of infection if massively disseminated. MβL's scaffold is designed to accommodate one or two zinc ions able to activate a nucleophilic hydroxide for the hydrolysis of the β-lactam ring. The role of zinc content on binding and reactive mechanism of action has been the subject of debate and still remains an open issue despite the large amount of data acquired. We report herein a study of the reaction pathway for binuclear CcrA from Bacteroides fragilis using density functional theory based quantum mechanicsmolecular mechanics dynamical modeling. CcrA is the prototypical binuclear enzyme belonging to B1 MβL family, which includes several harmful chromosomally-encoded and transferable enzymes. The involvement of a second zinc ion in the catalytic mechanism lowers the energetic barrier for β-lactam hydrolysis, preserving the essential binding features found in mononuclear B1 enzymes (BcII from Bacillus cereus) while providing a more efficient single-step mechanism. Overall, this study suggests that uptake of a second equivalent zinc ion is evolutionary favored.

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Probing the Role of Asp-120(81) of Metallo-β-lactamase (IMP-1) by Site-directed Mutagenesis, Kinetic Studies, and X-ray Crystallography

Abstract: Metallo-␤-lactamase IMP-1 is a di-Zn(II) metalloenzyme that efficiently hydrolyzes

Cited by 61 publications

References 48 publications

His224 Alters the R2 Drug Binding Site and Phe218 Influences the Catalytic Efficiency of the Metallo-β-Lactamase VIM-7

His224 Alters the R2 Drug Binding Site and Phe218 Influences the Catalytic Efficiency of the Metallo-β-Lactamase VIM-7

Structural Insights into the Subclass B3 Metallo-β-Lactamase SMB-1 and the Mode of Inhibition by the Common Metallo-β-Lactamase Inhibitor Mercaptoacetate

Role of Zinc Content on the Catalytic Efficiency of B1 Metallo β-Lactamases

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