scholarly journals P174E Substitution in GES-1 and GES-5 β-Lactamases Improves Catalytic Efficiency toward Carbapenems

2018 ◽  
Vol 62 (5) ◽  
Author(s):  
Alessandra Piccirilli ◽  
Paola Sandra Mercuri ◽  
Moreno Galleni ◽  
Massimiliano Aschi ◽  
André Matagne ◽  
...  
Keyword(s):  
Clavulanic Acid ◽  
Catalytic Efficiency ◽  
Md Simulations ◽  
Hydrolytic Activity ◽  
Cd Spectroscopy ◽  
Saturation Mutagenesis ◽  
Wild Type ◽  
Structural Content ◽  
Drastic Increase

ABSTRACT GES-type β-lactamases are a group of enzymes that have evolved their hydrolytic activity against carbapenems. In this study, the role of residue 174 inside the Ω-loop of GES-1 and GES-5 was investigated. GES-1 P174E and GES-5 P174E mutants, selected by site saturation mutagenesis, were purified and kinetically characterized. In comparison with GES-1 and GES-5 wild-type enzymes, GES-1 P174E and GES-5 P174E mutants exhibited lower k cat and k cat / K m values for cephalosporins and penicillins. Concerning carbapenems, GES-1 P174E shared higher k cat values but lower K m values than those calculated for GES-1. The GES-1 P174E and GES-5 P174E mutants showed high hydrolytic efficiency for imipenem, with k cat / K m values 100- and 660-fold higher, respectively, than those of GES-1. Clavulanic acid and tazobactam are good inhibitors for both GES-1 P174E and GES-5 P174E . Molecular dynamic (MD) simulations carried out for GES-1, GES-5, GES-1 P174E , and GES-5 P174E complexed with imipenem and meropenem have shown that mutation at position 174 induces a drastic increase of enzyme flexibility, in particular in the Ω-loop. The circular dichroism (CD) spectroscopy spectra of the four enzymes indicate that the P174E substitution in GES-1 and GES-5 does not affect the secondary structural content of the enzymes.

scholarly journals Enhanced production of Clavulanic acid by improving glycerol utilization using reporter-guided mutagenesis of an industrial Streptomyces clavuligerus strain

2021 ◽  
Author(s):  
Chang-Hun Shin ◽  
Hang Soo Cho ◽  
Hyung-Jin Won ◽  
Ho Jeong Kwon ◽  
Chan-Wha Kim ◽  
...  
Keyword(s):  
Clavulanic Acid ◽  
Wild Type Strain ◽  
Random Mutagenesis ◽  
Streptomyces Clavuligerus ◽  
Wild Type ◽  
Mutant Selection ◽  
Enhanced Production ◽  
Industrial Strains ◽  
Glycerol Utilization

Abstract Clavulanic acid (CA) produced by Streptomyces clavuligerus is a clinically important β-lactamase inhibitor. It is known that glycerol utilization can significantly improve cell growth and CA production of S. clavuligerus. We found that the industrial CA-producing S. clavuligerus strain OR generated by random mutagenesis consumes less glycerol than the wild-type strain; we then developed a mutant strain in which the glycerol utilization operon is overexpressed, as compared to the parent OR strain, through iterative random mutagenesis and reporter-guided selection. The CA production of the resulting S. clavuligerus ORUN strain was increased by approximately 31.3 per cent (5.21 ± 0.26 g/L) in a flask culture and 17.4 per cent (6.11 ± 0.36 g/L) in a fermenter culture, as compared to that of the starting OR strain. These results confirmed the important role of glycerol utilization in CA production and demonstrated that reporter-guided mutant selection is an efficient method for further improvement of randomly mutagenized industrial strains.

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 A 13C-NMR study of the role of Asn-155 in stabilizing the oxyanion of a subtilisin tetrahedral adduct

1997 ◽  
Vol 326 (3) ◽  
pp. 861-866 ◽  
Author(s):  
Timothy P. O'CONNELL ◽  
Regina M. DAY ◽  
Ekaterina V. TORCHILIN ◽  
William W. BACHOVCHIN ◽  
J. Paul G. MALTHOUSE
Keyword(s):  
13C Nmr ◽  
Chemical Shifts ◽  
Catalytic Efficiency ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Imidazolium Cation ◽  
Nmr Study ◽  
In The Wild ◽  
Main Factor

By removing one of the hydrogen-bond donors in the oxyanion hole of subtilisin BPN, we have been able to determine how it affects the catalytic efficiency of the enzyme and the pKa of the oxyanion formed in a choloromethane inhibitor derivative. Variant 8397 of subtilisin BPN contains five mutations which enhance its stability. Site-directed mutagenesis was used to prepare the N155A mutant of this variant. The catalytic efficiencies of wild-type and variant 8397 are similar, but replacing Asn-155 with alanine reduces catalytic efficiency approx. 300-fold. All three forms of subtilisin were alkylated using benzyloxycarbonylglycylglycyl[2-13C]phenylalanylchloromethane and examined by 13C-NMR. A single signal due to the 13C-enriched carbon was detected in all the derivatives and it was assigned to the hemiketal carbon of a tetrahedral adduct formed between the hydroxy group of Ser-221 and the inhibitor. This signal had chemical shifts in the range 98.3–103.6 p.p.m., depending on the pH. The titration shift of 4.7–4.8 p.p.m. was assigned to oxyanion formation. The oxyanion pKa values in the wild-type and 8397 variants were 6.92 and 7.00 respectively. In the N155A mutant of the 8397 variant the oxyanion pKa increased to 8.09. We explain why such a small increase is observed and we conclude that it is the interaction between the oxyanion and the imidazolium cation of the active-site histidine that is the main factor responsible for lowering the oxyanion pKa.

scholarly journals Expression of the Schwanniomyces occidentalis SWA2 amylase in Saccharomyces cerevisiae: role of N-glycosylation on activity, stability and secretion

1998 ◽  
Vol 329 (1) ◽  
pp. 65-71 ◽  
Author(s):  
Esther YÁÑEZ ◽  
A. Teresa CARMONA ◽  
Mercedes TIEMBLO ◽  
Antonio JIMÉNEZ ◽  
María FERNÁNDEZ-LOBATO
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Catalytic Efficiency ◽  
Extracellular Enzyme ◽  
Site Directed Mutagenesis ◽  
Activity Levels ◽  
Wild Type ◽  
Schwanniomyces Occidentalis ◽  
Type Protein ◽  
Wild Type Protein

The role of N-linked glycosylation on the biological activity of Schwanniomyces occidentalis SWA2 α-amylase, as expressed in Saccharomyces cerevisiae, was analysed by site-directed mutagenesis of the two potential N-glycosylation sites, Asn-134 and Asn-229. These residues were replaced by Ala or Gly individually or in various combinations and the effects on the activity, secretion and thermal stability of the enzyme were studied. Any Asn-229 substitution caused a drastic decrease in activity levels of the extracellular enzyme. In contrast, substitutions of Asn-134 had little or no effect. The use of antibodies showed that α-amylase was secreted in all the mutants tested, although those containing substitutions at Asn-229 seemed to have a lower rate of synthesis and/or higher degradation than the wild-type strain. α-Amylases with substitution at Asn-229 had a 2 kDa lower molecular mass than the wild-type protein, as did the wild-type protein itself after treatment with endoglycosidase F. These findings indicate that Asn-229 is the single glycosylated residue in SWA2. Thermostability analysis of both purified wild-type (T50 = 50 °C, where T50 is the temperature resulting in 50% loss of activity) and mutant enzymes indicated that removal of carbohydrate from the 229 position results in a decrease of approx. 3 °C in the T50 of the enzyme. The Gly-229 mutation does not change the apparent affinity of the enzyme for starch (Km) but decreases to 1/22 its apparent catalytic efficiency (kcat/Km). These results therefore indicate that glycosylation at the 229 position has an important role in the extracellular activity levels, kinetics and stability of the Sw. occidentalis SWA2 α-amylase in both its wild-type and mutant forms.

scholarly journals Substitutions at Position 105 in SHV Family β-Lactamases Decrease Catalytic Efficiency and Cause Inhibitor Resistance

2012 ◽  
Vol 56 (11) ◽  
pp. 5678-5686 ◽  
Author(s):  
Mei Li ◽  
Benjamin C. Conklin ◽  
Magdalena A. Taracila ◽  
Rebecca A. Hutton ◽  
Marion J. Skalweit
Keyword(s):  
Clavulanic Acid ◽  
Susceptibility Testing ◽  
Catalytic Efficiency ◽  
Wild Type ◽  
Oxyanion Hole ◽  
Content Type ◽  
Class A ◽  
Inhibitor Resistance ◽  
Structural Probes ◽  
Variant Residue

ABSTRACTAmbler position 105 in class A β-lactamases is implicated in resistance to clavulanic acid, although no clinical isolates with mutations at this site have been reported. We hypothesized that Y105 is important in resistance to clavulanic acid because changes in positioning of the inhibitor for ring oxygen protonation could occur. In addition, resistance to bicyclic 6-methylidene penems, which are interesting structural probes that inhibit all classes of serine β-lactamases with nanomolar affinity, might emerge with substitutions at position 105, especially with nonaromatic substitutions. All 19 variants of SHV-1 with variations at position 105 were prepared. Antimicrobial susceptibility testing showed thatEscherichia coliDH10B expressing Y105 variants retained activity against ampicillin, except for the Y105L variant, which was susceptible to all β-lactams, similar to the case for the host control strain. Several variants had elevated MICs to ampicillin-clavulanate. However, all the variants remained susceptible to piperacillin in combination with a penem inhibitor (MIC, ≤2/4 mg/liter). The Y105E, -F, -M, and -R variants demonstrated reduced catalytic efficiency toward ampicillin compared to the wild-type (WT) enzyme, which was caused by increasedKm. Clavulanic acid and penemKivalues were also increased for some of the variants, especially Y105E. Mutagenesis at position 105 in SHV yields mutants resistant to clavulanate with reduced catalytic efficiency for ampicillin and nitrocefin, similar to the case for the class A carbapenemase KPC-2. Our modeling analyses suggest that resistance is due to oxyanion hole distortion. Susceptibility to a penem inhibitor is retained although affinity is decreased, especially for the Y105E variant. Residue 105 is important to consider when designing new inhibitors.

scholarly journals Understanding the Role of R266K Mutation in Cystathionine β-Synthase (CBS) Enzyme: An in Silico Study

2021 ◽  
Author(s):  
Aashish Bhatt ◽  
Md. Ehesan Ali
Keyword(s):  
Hydrogen Bonding ◽  
In Silico ◽  
Density Functional ◽  
Experimental Studies ◽  
Salt Bridge ◽  
Md Simulations ◽  
Enzymatic Activities ◽  
Wild Type ◽  
Long Distance

<div>Human cystathionine β-synthase (hCBS) is a unique pyridoxal 5’-phosphate (PLP) dependent enzyme that catalyses the condensation reactions in the transsulfuration pathways. The specific role of Heme in the enzymatic activities has not yet been established, however, several experimental studies indicated the bi-directional communications between the Heme and PLP. Performing classical molecular dynamics (MD) simulations upon developing the necessary force field parameters for the cysteine and histidine bound hexa-coordinated Heme, we have investigated <i>In Silico</i> dynamical aspects of the bi-directional communications. Furthermore, we have investigated the comparative aspects of electron density overlap across the communicating pathways adopting the density functional theory (DFT) in conjunction with the hybrid exchange correlation functional for the CSB<sup>WT</sup> (wild-type) and CBS<sup>R266K</sup> (mutated) case. The atomistic dynamical simulations and subsequent explorations of the electronic structure not only confirm the reported observations but provide an in-depth mechanistic understating of how the non-covalent hydrogen bonding interactions with Cys52 control the such long-distance communication. Our study also provides a convincing answer to the reduced enzymatic activities in the R266K hCBS in comparison to the wild-type enzymes. We further realized that the difference in hydrogen-bonding patterns as well as salt-bridge interactions play the pivotal role in such long distant bi-directional communications.</div>

scholarly journals Kinetic Studies on CphA Mutants Reveal the Role of the P158-P172 Loop in Activity versus Carbapenems

2016 ◽  
Vol 60 (5) ◽  
pp. 3123-3126 ◽  
Author(s):  
Carlo Bottoni ◽  
Mariagrazia Perilli ◽  
Francesca Marcoccia ◽  
Alessandra Piccirilli ◽  
Cristina Pellegrini ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Catalytic Efficiency ◽  
Kinetic Studies ◽  
Zinc Ion ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
The Stability

ABSTRACTSite-directed mutagenesis of CphA indicated that prolines in the P158-P172 loop are essential for the stability and the catalytic activity of subclass B2 metallo-β-lactamases against carbapenems. The sequential substitution of proline led to a decrease of the catalytic efficiency of the variant compared to the wild-type (WT) enzyme but also to a higher affinity for the binding of the second zinc ion.

scholarly journals Characterization of the Novel CMT Enzyme TEM-154

2010 ◽  
Vol 55 (3) ◽  
pp. 1262-1265 ◽  
Author(s):  
Frédéric Robin ◽  
Julien Delmas ◽  
Elisabete Machado ◽  
Bernadette Bouchon ◽  
Luísa Peixe ◽  
...  
Keyword(s):  
Clavulanic Acid ◽  
Catalytic Efficiency ◽  
Hydrolytic Activity ◽  
European Countries ◽  
The Novel ◽  
Low Level ◽  
Extended Spectrum

ABSTRACTTEM-154, identified in Portugal in 2004, associated the substitutions observed in the extended-spectrum β-lactamase (ESBL) TEM-12 and in the inhibitor-resistant penicillinase (IRT) TEM-33. This enzyme exhibited hydrolytic activity against ceftazidime and a low level of resistance to clavulanic acid. Surprisingly, the substitution Met69Leu enhanced the catalytic efficiency of oxyimino β-lactams conferred by the substitution Arg164Ser. Its discovery confirms the dissemination of the complex mutant group of TEM enzymes in European countries.

scholarly journals Alteration of Leucine Aminopeptidase from Streptomyces septatus TH-2 to Phenylalanine Aminopeptidase by Site-Directed Mutagenesis

2005 ◽  
Vol 71 (11) ◽  
pp. 7229-7235 ◽  
Author(s):  
Jiro Arima ◽  
Yoshiko Uesugi ◽  
Masaki Iwabuchi ◽  
Tadashi Hatanaka
Keyword(s):  
Methyl Ester ◽  
Leucine Aminopeptidase ◽  
Hydrolytic Activity ◽  
Site Directed Mutagenesis ◽  
Saturation Mutagenesis ◽  
Side Chain ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Peptide Substrates ◽  
Hydrolytic Activities

ABSTRACT To tailor leucine aminopeptidase from Streptomyces septatus TH-2 (SSAP) to become a convenient biocatalyst, we are interested in Phe221 of SSAP, which is thought to interact with the side chain of the N-terminal residue of the substrate. By using saturation mutagenesis, the feasibility of altering the performance of SSAP was evaluated. The hydrolytic activities of 19 mutants were investigated using aminoacyl p-nitroanilide (pNA) derivatives as substrates. Replacement of Phe221 resulted in changes in the activities of all the mutants. Three of these mutants, F221G, F221A, and F221S, specifically hydrolyzed l-Phe-pNA, and F221I SSAP exhibited hydrolytic activity with l-Leu-pNA exceeding that of the wild type. Although the hydrolytic activities with peptide substrates decreased, the hydrolytic activities with amide and methyl ester substrates were proportional to the changes in the hydrolytic activities with pNA derivatives. Furthermore, based on a comparative kinetic study, the mechanism underlying the alteration in the preference of SSAP from leucine to phenylalanine is discussed.

scholarly journals Role of P1 residues Arg336 and Arg562 in the activated-Protein-C-catalysed inactivation of Factor VIIIa

2006 ◽  
Vol 396 (2) ◽  
pp. 355-362 ◽  
Author(s):  
Fatbardha Varfaj ◽  
Julie Neuberg ◽  
P. Vincent Jenkins ◽  
Hironao Wakabayashi ◽  
Philip J. Fay
Keyword(s):  
Protein C ◽  
Activated Protein C ◽  
Catalytic Efficiency ◽  
Wild Type ◽  
High Concentration ◽  
Subunit Dissociation ◽  
Factor Viiia ◽  
Km Value ◽  
Type Factor

APC (activated Protein C) inactivates human Factor VIIIa following cleavage at residues Arg336 and Arg562 within the A1 and A2 subunits respectively. The role of the P1 arginine in APC-catalysed inactivation of Factor VIIIa was examined by employing recombinant Factor VIIIa molecules where residues 336 and 562 were replaced with alanine and/or glutamine. Stably expressed Factor VIII proteins were activated by thrombin and resultant Factor VIIIa was reacted at high concentration with APC to minimize cofactor inactivation due to A2 subunit dissociation. APC cleaved wild-type Factor VIIIa at the A1 site with a rate ∼25-fold greater than that for the A2 site. A1 mutants R336A and R336Q were inactivated ∼9-fold slower than wild-type Factor VIIIa, whereas the A2 mutant R562A was inactivated ∼2-fold slower. No cleavage at the mutated sites was observed. Taken together, these results suggested that cleavage at the A1 site was the dominant mechanism for Factor VIIIa inactivation catalysed by the proteinase. On the basis of cleavage at Arg336, a Km value for wild-type Factor VIIIa of 102 nM was determined, and this value was significantly greater than Ki values (∼9–18 nM) obtained for an R336Q/R562Q Factor VIIIa. Furthermore, evaluation of a series of cluster mutants in the C-terminal region of the A1 subunit revealed a role for acidic residues in segment 341–345 in the APC-catalysed proteolysis of Arg336. Thus, while P1 residues contribute to catalytic efficiency, residues removed from these sites make a primary contribution to the overall binding of APC to Factor VIIIa.

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