Michiyoshi Nukaga

Functional Analysis of the Active Site of a Metallo-β-Lactamase Proliferating in Japan

ABSTRACT An R-plasmid-mediated metallo-β-lactamase was found inKlebsiella pneumoniae DK4 isolated in Japan in 1991. The nucleotide sequence of its structural gene revealed that the β-lactamase termed DK4 was identical to the IMP-1 metallo-β-lactamase which was mediated by a chromosomal gene ofSerratia marcescens TN9106 isolated in Japan in 1991 (E. Osano et al., Antimicrob. Agents Chemother. 38:71–78, 1994). The dose effect of DK4 β-lactamase production on the resistance levels indicated a significant contribution of the enzyme to bacterial resistance to all the β-lactams except monobactams. The enzymatic characteristics of the DK4 β-lactamase and its kinetic parameters for nine β-lactams were examined. The DK4 β-lactamase was confirmed to contain 2 mol of zinc per mol of enzyme protein. The apoenzyme that lacked the two zincs was structurally unstable, and the activities of only 30% of the apoenzyme molecules could be restored by the addition of 1 mM zinc sulfate. The substitution of five conserved histidines (His28, His86, His88, His149, His210) and a cysteine (Cys168) for an alanine indicated that His86, His88, and His149 served as ligands to one of the zincs and that Cys168 played a role as a ligand to the second zinc. Both zinc molecules contribute to the enzymatic process. Mutant enzymes that lack only one of these retained some activity. Additionally, a conserved aspartic acid at position 90 was replaced by asparagine. This mutant enzyme showed an approximately 1,000 times lower kcat value for cephalothin than that of the wild-type enzyme but retained the two zincs even after dialysis against zinc-free buffer. The observed effect of pH on the activity suggested that Asp90 functions as a general base in the enzymatic process.

Insights into β-Lactamases from Burkholderia Species, Two Phylogenetically Related yet Distinct Resistance Determinants

Background: Resistance to β-lactams in Burkholderia is mediated by different β-lactamases (e.g. PenA and PenI). Results: PenA from B. multivorans is a carbapenemase, and PenI from B. pseudomallei is an extended-spectrum enzyme. Conclusion: Subtle changes within the active site of β-lactamases result in major phenotypic changes. Significance: Future antibiotic design must consider the distinctive phenotypes of PenA and PenI β-lactamases. Burkholderia cepacia complex and Burkholderia pseudomallei are opportunistic human pathogens. Resistance to β-lactams among Burkholderia spp. is attributable to expression of β-lactamases (e.g. PenA in B. cepacia complex and PenI in B. pseudomallei). Phylogenetic comparisons reveal that PenA and PenI are highly related. However, the analyses presented here reveal that PenA is an inhibitor-resistant carbapenemase, most similar to KPC-2 (the most clinically significant serine carbapenemase), whereas PenI is an extended spectrum β-lactamase. PenA hydrolyzes β-lactams with kcat values ranging from 0.38 ± 0.04 to 460 ± 46 s−1 and possesses high kcat/kinact values of 2000, 1500, and 75 for β-lactamase inhibitors. PenI demonstrates the highest kcat value for cefotaxime of 9.0 ± 0.9 s−1. Crystal structure determination of PenA and PenI reveals important differences that aid in understanding their contrasting phenotypes. Changes in the positioning of conserved catalytic residues (e.g. Lys-73, Ser-130, and Tyr-105) as well as altered anchoring and decreased occupancy of the deacylation water explain the lower kcat values of PenI. The crystal structure of PenA with imipenem docked into the active site suggests why this carbapenem is hydrolyzed and the important role of Arg-220, which was functionally confirmed by mutagenesis and biochemical characterization. Conversely, the conformation of Tyr-105 hindered docking of imipenem into the active site of PenI. The structural and biochemical analyses of PenA and PenI provide key insights into the hydrolytic mechanisms of β-lactamases, which can lead to the rational design of novel agents against these pathogens.

Thrombin‐stimulated proliferation is mediated by endothelin‐1 in cultured rat gingival fibroblasts

Endothelin‐1 (ET‐1) appears to be involved in drug‐induced proliferation of gingival fibroblasts. Thrombin induces proliferation of human gingival fibroblasts via protease‐activated receptor 1 (PAR1). In this study, using cultured rat gingival fibroblasts, we investigated whether thrombin‐induced proliferation of gingival fibroblasts is mediated by ET‐1. Thrombin‐induced proliferation (0.05–2.5 U/mL). Proliferation was also induced by a PAR1‐specific agonist (TFLLR‐NH2, 0.1–30 μm), but not by a PAR2‐specific agonist (SLIGRL‐NH2). Thrombin (2.5 U/mL) induced an increase in immunoreactive ET‐1 expression, which was inhibited by cycloheximide (10 μg/mL), and an increase in preproET‐1 mRNA expression, as assessed by reverse transcription polymerase chain reaction. TFLLR‐NH2 increased ET‐1 release into the culture medium in both a concentration (0.01–10 μm)‐ and time (6–24 h)‐dependent manner, as assessed by solid phase sandwich enzyme‐linked immunosorbent assay. The thrombin (2.5 U/mL)‐induced proliferation was inhibited by a PAR1‐selective inhibitor, SCH79797 (0.1 μm) and an ETA antagonist, BQ‐123 (1 μm), but not by an ETB antagonist, BQ‐788 (1 μm). These findings suggest that thrombin, acting via PAR1, induced proliferation of cultured rat gingival fibroblasts that was mediated by ET‐1 acting via ETA.

Inhibition of Class D β-Lactamases by Acyl Phosphates and Phosphonates

ABSTRACT The susceptibility of typical class D β-lactamases to inhibition by acyl phosph(on)ates has been determined. To a large degree, these class D enzymes behaved very similarly to the class A TEM β-lactamase towards these reagents. Dibenzoyl phosphate stood out in both cases as a lead compound towards a new class of effective inhibitors.

Structural and computational study on inhibitory compounds for endonuclease activity of influenza virus polymerase.

Seasonal epidemics and occasional pandemics caused by influenza viruses are global threats to humans. Since the efficacy of currently approved drugs is limited by the emerging resistance of the viruses, the development of new antiviral drugs is still demanded. Endonuclease activity, which lies in the influenza polymerase acidic protein N-terminal domain (PA(N)), is a potent target for novel antiviral agents. Here, we report the identification of some novel inhibitors for PA(N) endonuclease activity. The binding mode of one of the inhibitory compounds to PA(N) was investigated in detail by means of X-ray crystal structure analysis and molecular dynamics (MD) simulation. It was observed in the crystal structure that three molecules of the same kind of inhibitor were bound to one PA(N). One of the three molecules is located at the active site and makes a chelation to metal ions. Another molecule is positioned at the space adjacent to the metal-chelated site. The other molecule is located at a site slightly apart from the metal-chelated site, causing a conformational change of Arg124. The last binding site was not observed in previous crystallographic studies. Hence, the stability of inhibitor binding was examined by performing 100-ns MD simulation. During the MD simulation, the three inhibitor molecules fluctuated at the respective binding sites at different amplitudes, while all of the molecules maintained interactions with the protein. Molecular mechanics/generalized Born surface area (MM/GBSA) analysis suggested that the molecule in the last binding site has a higher affinity than the others. Structural information obtained in this study will provide a hint for designing and developing novel potent agents against influenza viruses.

Interaction of oxyimino beta-lactams with a class C beta-lactamase and a mutant with a spectrum extended to beta-lactams.

The class C beta-lactamase of Citrobacter freundii GN346 is a typical cephalosporinase comprising 361 amino acids, and substitution of the glutamic acid at position 219 in the enzyme by lysine was previously shown to broaden its substrate spectrum to oxyimino beta-lactams (K. Tsukamoto, R. Ohno, and T. Sawai, J. Bacteriol. 172:4348-4351, 1990). To clarify this spectrum extension from the kinetic point of view, the interactions of cefuroxime, ceftazidime, and aztreonam with the wild-type and mutant enzymes were analyzed. In addition to aztreonam, known as a progressive inhibitor of class C beta-lactamases, cefuroxime and ceftazidime were found to act as progressive inhibitors of the wild-type enzyme. On the other hand, only aztreonam showed weak progressive inhibition of the mutant enzyme. On the basis of kinetic parameters, a minimum scheme for interaction of the oxyimino beta-lactams with the wild-type enzyme was proposed, and the rate-limiting step of the hydrolysis of unfavorable substrates was indicated to be conversion of the stable acyl-enzyme intermediate to the unstable intermediate. In aztreonam hydrolysis by the mutant enzyme, the reaction rate at the rate-limiting step was 2,000 times that of the wild-type enzyme. These results indicate that the mutation at position 219 disturbs the stabilization of the stable intermediate.

Two Distinctive Binding Modes of Endonuclease Inhibitors to the N-Terminal Region of Influenza Virus Polymerase Acidic Subunit

Influenza viruses are global threat to humans, and the development of new antiviral agents are still demanded to prepare for pandemics and to overcome the emerging resistance to the current drugs. Influenza polymerase acidic protein N-terminal domain (PAN) has endonuclease activity and is one of the appropriate targets for novel antiviral agents. First, we performed X-ray cocrystal analysis on the complex structures of PAN with two endonuclease inhibitors. The protein crystallization and the inhibitor soaking were done at pH 5.8. The binding modes of the two inhibitors were different from a common binding mode previously reported for the other influenza virus endonuclease inhibitors. We additionally clarified the complex structures of PAN with the same two endonuclease inhibitors at pH 7.0. In one of the crystal structures, an additional inhibitor molecule, which chelated to the two metal ions in the active site, was observed. On the basis of the crystal structures at pH 7.0, we carried out 100 ns molecular dynamics (MD) simulations for both of the complexes. The analysis of simulation results suggested that the binding mode of each inhibitor to PAN was stable in spite of the partial deviation of the simulation structure from the crystal one. Furthermore, crystal structure analysis and MD simulation were performed for PAN in complex with an inhibitor, which was already reported to have a high compound potency for comparison. The findings on the presence of multiple binding sites at around the PAN substrate-binding pocket will provide a hint for enhancing the binding affinity of inhibitors.