scholarly journals Biochemical Characterization of Metallo-β-Lactamase VIM-11 from a Pseudomonas aeruginosa Clinical Strain

2008 ◽  
Vol 52 (6) ◽  
pp. 2250-2252 ◽  
Author(s):  
Patricia Marchiaro ◽  
Pablo E. Tomatis ◽  
María A. Mussi ◽  
Fernando Pasteran ◽  
Alejandro M. Viale ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biochemical Characterization ◽  
Catalytic Efficiency ◽  
Clinical Strain

ABSTRACT A detailed biochemical characterization of the Pseudomonas aeruginosa VIM-11 metallo-β-lactamase (MβL) is reported. The only substitution differentiating VIM-11 from VIM-2 (N165S) promoted a slightly improved catalytic efficiency of the former on 3 out of 12 substrates, notably the bulky cephalosporins. Thus, MβL-mediated resistance also may be modulated by remote mutations.

scholarly journals Characterization of an Arginine:Pyruvate Transaminase in Arginine Catabolism of Pseudomonas aeruginosa PAO1

2007 ◽  
Vol 189 (11) ◽  
pp. 3954-3959 ◽  
Author(s):  
Zhe Yang ◽  
Chung-Dar Lu
Keyword(s):  
Pseudomonas Aeruginosa ◽  
High Performance ◽  
Biochemical Characterization ◽  
Physiological Function ◽  
Catalytic Efficiency ◽  
Kinetic Mechanism ◽  
Arginine Catabolism ◽  
Ping Pong ◽  
Coupled Reaction

ABSTRACT The arginine transaminase (ATA) pathway represents one of the multiple pathways for l-arginine catabolism in Pseudomonas aeruginosa. The AruH protein was proposed to catalyze the first step in the ATA pathway, converting the substrates l-arginine and pyruvate into 2-ketoarginine and l-alanine. Here we report the initial biochemical characterization of this enzyme. The aruH gene was overexpressed in Escherichia coli, and its product was purified to homogeneity. High-performance liquid chromatography and mass spectrometry (MS) analyses were employed to detect the presence of the transamination products 2-ketoarginine and l-alanine, thus demonstrating the proposed biochemical reaction catalyzed by AruH. The enzymatic properties and kinetic parameters of dimeric recombinant AruH were determined by a coupled reaction with NAD+ and l-alanine dehydrogenase. The optimal activity of AruH was found at pH 9.0, and it has a novel substrate specificity with an order of preference of Arg > Lys > Met > Leu > Orn > Gln. With l-arginine and pyruvate as the substrates, Lineweaver-Burk plots of the data revealed a series of parallel lines characteristic of a ping-pong kinetic mechanism with calculated V max and k cat values of 54.6 ± 2.5 μmol/min/mg and 38.6 ± 1.8 s−1. The apparent Km and catalytic efficiency (k cat/Km ) were 1.6 ± 0.1 mM and 24.1 mM−1 s−1 for pyruvate and 13.9 ± 0.8 mM and 2.8 mM−1 s−1 for l-arginine. When l-lysine was used as the substrate, MS analysis suggested Δ1-piperideine-2-carboxylate as its transamination product. These results implied that AruH may have a broader physiological function in amino acid catabolism.

scholarly journals Molecular and Biochemical Characterization of OXA-45, an Extended-Spectrum Class 2d′ β-Lactamase in Pseudomonas aeruginosa

2003 ◽  
Vol 47 (9) ◽  
pp. 2859-2863 ◽  
Author(s):  
Mark A. Toleman ◽  
Kenneth Rolston ◽  
Ronald N. Jones ◽  
Timothy R. Walsh
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biochemical Characterization ◽  
Polymyxin B ◽  
Clinical Strain ◽  
Surveillance Program ◽  
Extended Spectrum ◽  
Class D ◽  
Antimicrobial Surveillance ◽  
Serovar Typhimurium

ABSTRACT As part of the CANCER Antimicrobial Surveillance Program in North America, a clinical strain of Pseudomonas aeruginosa, strain 07-406, isolated in Texas was found to be resistant to all antimicrobials except polymyxin B. Genetic analysis of this isolate identified two unique extended-spectrum β-lactamase genes. One, bla VIM-7, encoded a metallo-β-lactamase (unpublished data), and the other, bla OXA-45, described here, encoded a class D extended-spectrum β-lactamase. bla OXA-45 was isolated on a Sau3A1 genomic fragment of 1.8 kb and encodes a protein of 264 amino acids with the highest identities to OXA-18 (65.9%), OXA-9 (42.8%), OXA-22 (40.2%), OXA-12 (38.6%), and OXA-29 (35.2%) but weak identities with other class D β-lactamases. bla OXA-45 was found to be harbored on a 24-kb plasmid in a region that displays high identities with a section of the 43-kb genomic island of Salmonella enterica serovar Typhimurium DT104. Biochemically OXA-45 is most similar to OXA-18 in its substrate profile and inhibition by clavulanic acid and is a member of the 2d′ class of β-lactamases.

scholarly journals Structural and biochemical characterization of the environmental MBLs MYO-1, ECV-1 and SHD-1

2020 ◽  
Vol 75 (9) ◽  
pp. 2554-2563 ◽  
Author(s):  
Christopher Fröhlich ◽  
Vidar Sørum ◽  
Sandra Huber ◽  
Ørjan Samuelsen ◽  
Fanny Berglund ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Homology Modelling ◽  
Catalytic Efficiency ◽  
Hydrolytic Activity ◽  
Human Pathogens ◽  
E Coli ◽  
X Ray Crystallography ◽  
Environmental Reservoir

Abstract Background MBLs form a large and heterogeneous group of bacterial enzymes conferring resistance to β-lactam antibiotics, including carbapenems. A large environmental reservoir of MBLs has been identified, which can act as a source for transfer into human pathogens. Therefore, structural investigation of environmental and clinically rare MBLs can give new insights into structure–activity relationships to explore the role of catalytic and second shell residues, which are under selective pressure. Objectives To investigate the structure and activity of the environmental subclass B1 MBLs MYO-1, SHD-1 and ECV-1. Methods The respective genes of these MBLs were cloned into vectors and expressed in Escherichia coli. Purified enzymes were characterized with respect to their catalytic efficiency (kcat/Km). The enzymatic activities and MICs were determined for a panel of different β-lactams, including penicillins, cephalosporins and carbapenems. Thermostability was measured and structures were solved using X-ray crystallography (MYO-1 and ECV-1) or generated by homology modelling (SHD-1). Results Expression of the environmental MBLs in E. coli resulted in the characteristic MBL profile, not affecting aztreonam susceptibility and decreasing susceptibility to carbapenems, cephalosporins and penicillins. The purified enzymes showed variable catalytic activity in the order of <5% to ∼70% compared with the clinically widespread NDM-1. The thermostability of ECV-1 and SHD-1 was up to 8°C higher than that of MYO-1 and NDM-1. Using solved structures and molecular modelling, we identified differences in their second shell composition, possibly responsible for their relatively low hydrolytic activity. Conclusions These results show the importance of environmental species acting as reservoirs for MBL-encoding genes.

scholarly journals Molecular and Biochemical Characterization of SHV-56, a Novel Inhibitor-Resistant β-Lactamase from Klebsiella pneumoniae

2008 ◽  
Vol 52 (10) ◽  
pp. 3792-3794 ◽  
Author(s):  
Véronique Dubois ◽  
Laurent Poirel ◽  
François Demarthe ◽  
Corinne Arpin ◽  
Laure Coulange ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Biochemical Characterization ◽  
Clinical Strain ◽  
Chromosomal Gene ◽  
Inhibitor Resistance ◽  
Critical Site ◽  
Single Substitution

ABSTRACT A clinical strain of Klebsiella pneumoniae was found to possess the chromosomal gene bla SHV-56, encoding a new inhibitor-resistant β-lactamase with a pI of 7.6. SHV-56 is derived from SHV-11 by the single substitution K234R. This mutation therefore evidences a new critical site for inhibitor resistance among SHV enzymes.

scholarly journals Biochemical and Structural Characterization of the Subclass B1 Metallo-β-Lactamase VIM-4

2010 ◽  
Vol 55 (3) ◽  
pp. 1248-1255 ◽  
Author(s):  
Patricia Lassaux ◽  
Daouda A. K. Traoré ◽  
Elodie Loisel ◽  
Adrien Favier ◽  
Jean-Denis Docquier ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Thermal Stability ◽  
Pseudomonas Aeruginosa ◽  
Structural Characterization ◽  
Catalytic Efficiency ◽  
X Ray ◽  
Stabilizing Effect ◽  
Loop 2

ABSTRACTThe metallo-β-lactamase VIM-4, mainly found inPseudomonas aeruginosaorAcinetobacter baumannii, was produced inEscherichia coliand characterized by biochemical and X-ray techniques. A detailed kinetic study performed in the presence of Zn2+at concentrations ranging from 0.4 to 100 μM showed that VIM-4 exhibits a kinetic profile similar to the profiles of VIM-2 and VIM-1. However, VIM-4 is more active than VIM-1 against benzylpenicillin, cephalothin, nitrocefin, and imipenem and is less active than VIM-2 against ampicillin and meropenem. The crystal structure of the dizinc form of VIM-4 was solved at 1.9 Å. The sole difference between VIM-4 and VIM-1 is found at residue 228, which is Ser in VIM-1 and Arg in VIM-4. This substitution has a major impact on the VIM-4 catalytic efficiency compared to that of VIM-1. In contrast, the differences between VIM-2 and VIM-4 seem to be due to a different position of the flapping loop and two substitutions in loop 2. Study of the thermal stability and the activity of the holo- and apo-VIM-4 enzymes revealed that Zn2+ions have a pronounced stabilizing effect on the enzyme and are necessary for preserving the structure.

scholarly journals Functional and Structural Characterization of OXA-935, a Novel OXA-10-family β-lactamase from Pseudomonas aeruginosa

2021 ◽  
Author(s):  
Nathan B Pincus ◽  
Monica Rosas-Lemus ◽  
Samuel WM Gatesy ◽  
Ludmilla A. Shuvalova ◽  
Joseph Brunzelle ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Clonal Complex ◽  
Genomic Analysis ◽  
Catalytic Efficiency ◽  
Multidrug Resistant ◽  
Extended Spectrum ◽  
The Impact ◽  
Lactamase Inhibitor

Resistance to antipseudomonal penicillins and cephalosporins is often driven by the overproduction of the intrinsic β-lactamase AmpC. However, OXA-10-family β-lactamases are a rich source of resistance in Pseudomonas aeruginosa. OXA β-lactamases have a propensity for mutation leading to extended spectrum cephalosporinase and carbapenemase activity. In this study, we identified isolates from a subclade of the multidrug-resistant (MDR) high risk clonal complex CC446 with resistance to ceftazidime. Genomic analysis revealed that these isolates harbored a plasmid containing a novel allele of blaOXA-10, named blaOXA-935, which was predicted to produce an OXA-10 variant with two amino acid substitutions: an aspartic acid instead of glycine at position 157 and a serine instead of phenylalanine at position 153. The G157D mutation, present in OXA-14, is associated with resistance to ceftazidime. Deletion of blaOXA-935 restored sensitivity to ceftazidime and susceptibility profiling of P. aeruginosa laboratory strains expressing blaOXA-935 revealed that OXA-935 conferred ceftazidime resistance. To better understand the impact of the variant amino acids, we determined the crystal structures of OXA-14 and OXA-935. In contrast, both monomers of OXA-935 were decarbamylated at K70, and the F153S mutation conferred increased flexibility to the omega (Ω) loop. Compared to OXA-14, the catalytic efficiency of OXA-935 for nitrocefin was significantly reduced. Amino acid changes that confer extended spectrum cephalosporinase activity to OXA-10-family β-lactamases are concerning given rising reliance on novel β-lactam/β-lactamase inhibitor combinations such as ceftolozane-tazobactam and ceftazidime-avibactam to treat MDR P. aeruginosa infections.

scholarly journals Genetic and Biochemical Characterization of an Acquired Subgroup B3 Metallo-β-Lactamase Gene,blaAIM-1, and Its Unique Genetic Context in Pseudomonas aeruginosa from Australia

2012 ◽  
Vol 56 (12) ◽  
pp. 6154-6159 ◽  
Author(s):  
Dongeun Yong ◽  
Mark A. Toleman ◽  
Jan Bell ◽  
Brett Ritchie ◽  
Rachael Pratt ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Southern Hybridization ◽  
Biochemical Characterization ◽  
Gene Bank ◽  
Clinical Isolates ◽  
Gram Negative ◽  
Content Type ◽  
Lactamase Gene ◽  
Genetic Context

ABSTRACTThree clinicalPseudomonas aeruginosaisolates (WCH2677, WCH2813, and WCH2837) isolated from the Women's and Children's Hospital, Adelaide, Australia, produced a metallo-β-lactamase (MBL)-positive Etest result. All isolates were PCR negative for known MBL genes. A gene bank was created, and an MBL gene, designatedblaAIM-1, was cloned and fully characterized. The encoded enzyme, AIM-1, is a group B3 MBL that has the highest level of identity to THIN-B and L1. It is chromosomal and flanked by two copies (one intact and one truncated) of an ISCRelement, ISCR15. Southern hybridization studies indicated the movement of both ISCR15andblaAIM-1within the three different clinical isolates. AIM-1 hydrolyzes most β-lactams, with the exception of aztreonam and, to a lesser extent, ceftazidime; however, it possesses significantly higherkcatvalues for cefepime and carbapenems than most other MBLs. AIM-1 was the first mobile group B3 enzyme detected and signals further problems for already beleaguered antimicrobial regimes to treat seriousP. aeruginosaand other Gram-negative infections.

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