Biochemical and Structural Characterization of OXA-405, an OXA-48 Variant with Extended-Spectrum β-Lactamase Activity

OXA-48-producing Enterobacterales have now widely disseminated globally. A sign of their extensive spread is the identification of an increasing number of OXA-48 variants. Among them, three are particularly interesting, OXA-163, OXA-247 and OXA-405, since they have lost carbapenem activities and gained expanded-spectrum cephalosporin hydrolytic activity subsequent to a four amino-acid (AA) deletion in the β5–β6 loop. We investigated the mechanisms responsible for substrate specificity of OXA-405. Kinetic parameters confirmed that OXA-405 has a hydrolytic profile compatible with an ESBL (hydrolysis of expanded spectrum cephalosporins and susceptibility to class A inhibitors). Molecular modeling techniques and 3D structure determination show that the overall dimeric structure of OXA-405 is very similar to that of OXA-48, except for the β5–β6 loop, which is shorter for OXA-405, suggesting that the length of the β5–β6 loop is critical for substrate specificity. Covalent docking with selected substrates and molecular dynamics simulations evidenced the structural changes induced by substrate binding, as well as the distribution of water molecules in the active site and their role in substrate hydrolysis. All this data may represent the structural basis for the design of new and efficient class D inhibitors.

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An instrumented sample holder for time-lapse microtomography measurements of snow under advective airflow

Geoscientific Instrumentation Methods and Data Systems ◽

10.5194/gi-3-179-2014 ◽

2014 ◽

Vol 3 (2) ◽

pp. 179-185 ◽

Cited By ~ 7

Author(s):

P. P. Ebner ◽

S. A. Grimm ◽

M. Schneebeli ◽

A. Steinfeld

Keyword(s):

Structural Changes ◽

Time Lapse ◽

Temperature Gradients ◽

Sample Holder ◽

Experimental Characterization ◽

Computational Fluid Dynamics Simulations ◽

Dynamics Simulations ◽

Snow Samples

Abstract. An instrumented sample holder was developed for time-lapse microtomography of snow samples to enable in situ nondestructive spatial and temporal measurements under controlled advective airflows, temperature gradients, and air humidities. The design was aided by computational fluid dynamics simulations to evaluate the airflow uniformity across the snow sample. Morphological and mass transport properties were evaluated during a 4-day test run. This instrument allows the experimental characterization of metamorphism of snow undergoing structural changes with time.

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Structural Basis for Substrate Specificity and Carbapenemase Activity of OXA-48 Class D β-Lactamase

ACS Infectious Diseases ◽

10.1021/acsinfecdis.9b00304 ◽

2019 ◽

Vol 6 (2) ◽

pp. 261-271 ◽

Cited By ~ 2

Author(s):

Afroza Akhtar ◽

Orville A. Pemberton ◽

Yu Chen

Keyword(s):

Substrate Specificity ◽

Structural Basis ◽

Class D

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A novel endo-β-1,4-xylanase GH30 from Dickeya dadantii DCE-01: Clone, expression, characterization, and ramie biological degumming function

Textile Research Journal ◽

10.1177/0040517517748511 ◽

2017 ◽

Vol 89 (4) ◽

pp. 463-472 ◽

Cited By ~ 3

Author(s):

Ruijun Wang ◽

Zhengchu Liu ◽

Lifeng Cheng ◽

Shengwen Duan ◽

Xiangyuan Feng ◽

...

Keyword(s):

Biochemical Characterization ◽

Ph Value ◽

Hydrolytic Activity ◽

Glycoside Hydrolase Family ◽

Dickeya Dadantii ◽

Beechwood Xylan ◽

Expression Characterization ◽

Hydrolysis Of ◽

Hydrolase Family

Xylanase plays an important role in the hydrolysis of hemicellulose and has gained much attention in the field of biological degumming. The research for xylanases with cellulase-free and high activity for biological degumming has intensified in recent years. In the present research, heterologous expression of a novel endo-β-1,4-xylanase (GH30) from Dickeya dadantii DCE-01 in Escherichia coli BL21 (DE3) was reported. Biochemical characterization of the enzyme and a potential application in ramie biological degumming was discussed. The results showed that the xylanase gene consists of 1251 nucleotides, belonging to glycoside hydrolase family 30 (GH30). The optimal activity of the xylanase was observed at 50℃ and a pH value of 6.4. The Km and Vmax values for beechwood xylan were 14.25 mg/mL and 296.6 μmol/mg, respectively. The catalytic activity was enhanced by addition of 1 mM Cu2+, Ca2+, Mg2+, and K+. The recombinant enzyme was specific for xylan substrates. The enzyme exhibited hydrolytic activity toward ramie hemicellulose. The recombinant xylanase could be effectively applied to ramie degumming.

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Moving toward generalizable NZ-1 labeling for 3D structure determination with optimized epitope-tag insertion

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798321002527 ◽

2021 ◽

Vol 77 (5) ◽

pp. 645-662

Author(s):

Risako Tamura-Sakaguchi ◽

Rie Aruga ◽

Mika Hirose ◽

Toru Ekimoto ◽

Takuya Miyake ◽

...

Keyword(s):

Complex Formation ◽

Structure Determination ◽

Structural Changes ◽

Insertion Site ◽

3D Structure ◽

Binding Pocket ◽

Crystallographic Analysis ◽

Stable Complex ◽

X Ray Crystallography ◽

Dynamics Simulations

Antibody labeling has been conducted extensively for structure determination using both X-ray crystallography and electron microscopy (EM). However, establishing target-specific antibodies is a prerequisite for applying antibody-assisted structural analysis. To expand the applicability of this strategy, an alternative method has been developed to prepare an antibody complex by inserting an exogenous epitope into the target. It has already been demonstrated that the Fab of the NZ-1 monoclonal antibody can form a stable complex with a target containing a PA12 tag as an inserted epitope. Nevertheless, it was also found that complex formation through the inserted PA12 tag inevitably caused structural changes around the insertion site on the target. Here, an attempt was made to improve the tag-insertion method, and it was consequently discovered that an alternate tag (PA14) could replace various loops on the target without inducing large structural changes. Crystallographic analysis demonstrated that the inserted PA14 tag adopts a loop-like conformation with closed ends in the antigen-binding pocket of the NZ-1 Fab. Due to proximity of the termini in the bound conformation, the more optimal PA14 tag had only a minor impact on the target structure. In fact, the PA14 tag could also be inserted into a sterically hindered loop for labeling. Molecular-dynamics simulations also showed a rigid structure for the target regardless of PA14 insertion and complex formation with the NZ-1 Fab. Using this improved labeling technique, negative-stain EM was performed on a bacterial site-2 protease, which enabled an approximation of the domain arrangement based on the docking mode of the NZ-1 Fab.

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Characterization of a chymotrypsin-like hydrolytic activity in the opossum kidney cell

Biochemistry and Cell Biology ◽

10.1139/o94-023 ◽

1994 ◽

Vol 72 (3-4) ◽

pp. 157-162 ◽

Cited By ~ 4

Author(s):

Makoto Arao ◽

Toru Yamaguchi ◽

Toshitsugu Sugimoto ◽

Masaaki Fukase ◽

Kazuo Chihara

Keyword(s):

Parathyroid Hormone ◽

Molecular Mass ◽

Hydrolytic Activity ◽

High Molecular Mass ◽

Phenylmethylsulfonyl Fluoride ◽

Opossum Kidney ◽

Ok Cells ◽

Opossum Kidney Cell ◽

Hydrolysis Of

To characterize a chymotrypsin-like hydrolytic activity in the cell surface membranes of intact opossum kidney (OK) cells, we partially purified a protease from the membrane fractions of OK cells using Suc-Leu-Leu-Val-Tyr-MCA (Sue, succinyl; MCA, 4-methylcoumaryl-7-amide), a synthetic substrate for chymotrypsin, as the substrate. The semipure enzyme showed seryl chymotrypsin-like characteristics such as preferential hydrolysis of Suc-Leu-Leu-Val-Tyr-MCA and inhibition by phenylmethylsulfonyl fluoride, diisopropylfluorophosphate, and chymostatin. However, it clearly differed from α-chymotrypsin in its weak ability to hydrolyze Suc-Ala-Ala-Pro-Phe-MCA and in its high molecular mass (250–300 kDa). The enzyme also had an endopeptidase-like activity in that it cleaved human parathyroid hormone(1–84) at the Leu(37)-Gly(38) and Arg(52)-Lys(53) bonds. These results suggest that a high molecular mass chymotrypsin-like endopeptidase with unique characters is present in the membrane fractions of OK cells.Key words: opossum kidney, parathyroid hormone, chymotrypsin, endopeptidase.

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Structural Basis of Reduced Susceptibility to Ceftazidime-Avibactam and Cefiderocol in Enterobacter cloacae Due to AmpC R2 Loop Deletion

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00198-20 ◽

2020 ◽

Vol 64 (7) ◽

Cited By ~ 1

Author(s):

Akito Kawai ◽

Christi L. McElheny ◽

Alina Iovleva ◽

Ellen G. Kline ◽

Nicolas Sluis-Cremer ◽

...

Keyword(s):

Structural Changes ◽

Enterobacter Cloacae ◽

Multidrug Resistant ◽

Structural Basis ◽

Clinical Strain ◽

Resistant Bacteria ◽

Single Mutation ◽

Close Monitoring ◽

Content Type ◽

Hydrolysis Of

ABSTRACT Ceftazidime-avibactam and cefiderocol are two of the latest generation β-lactam agents that possess expanded activity against highly drug-resistant bacteria, including carbapenem-resistant Enterobacterales. Here, we show that structural changes in AmpC β-lactamases can confer reduced susceptibility to both agents. A multidrug-resistant Enterobacter cloacae clinical strain (Ent385) was found to be resistant to ceftazidime-avibactam and cefiderocol without prior exposure to either agent. The AmpC β-lactamase of Ent385 (AmpCEnt385) contained an alanine-proline deletion at positions 294 and 295 (A294_P295del) in the R2 loop. AmpCEnt385 conferred reduced susceptibility to ceftazidime-avibactam and cefiderocol when cloned into Escherichia coli TOP10. Purified AmpCEnt385 showed increased hydrolysis of ceftazidime and cefiderocol compared to AmpCEnt385Rev, in which the deletion was reverted. Comparisons of crystal structures of AmpCEnt385 and AmpCP99, the canonical AmpC of E. cloacae complex, revealed that the two-residue deletion in AmpCEnt385 induced drastic structural changes of the H-9 and H-10 helices and the R2 loop, which accounted for the increased hydrolysis of ceftazidime and cefiderocol. The potential for a single mutation in ampC to confer reduced susceptibility to both ceftazidime-avibactam and cefiderocol requires close monitoring.

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Oncogenic G12D mutation alters local conformations and dynamics of K-Ras

10.1101/178483 ◽

2017 ◽

Author(s):

Sezen Vatansever ◽

Burak Erman ◽

Zeynep H. Gümüş

Keyword(s):

Structural Changes ◽

Structural Basis ◽

Local Dynamic ◽

Wild Type ◽

Local Conformations ◽

Dynamics Simulations ◽

Direct Inhibitors ◽

Detailed Picture ◽

Inactive Protein ◽

Prevalent Mutation

AbstractK-Ras is the most frequently mutated oncoprotein in human cancers, and G12D is its most prevalent mutation. To understand how G12D mutation impacts K-Ras function, we need to understand how it alters the regulation of its dynamics. Here, we present local changes in K-Ras structure, conformation and dynamics upon G12D mutation, from long-timescale Molecular Dynamics simulations of active (GTP-bound) and inactive (GDP-bound) forms of wild-type and mutant K-Ras, with an integrated investigation of atomistic-level changes, local conformational shifts and correlated residue motions. Our results reveal that the local changes in K-Ras are specific to bound nucleotide (GTP or GDP), and we provide a structural basis for this. Specifically, we show that G12D mutation causes a shift in the population of local conformational states of K-Ras, especially in Switch-II (SII) and α3-helix regions, in favor of a conformation that is associated with a catalytically impaired state through structural changes; it also causes SII motions to anti-correlate with other regions. This detailed picture of G12D mutation effects on the local dynamic characteristics of both active and inactive protein helps enhance our understanding of local K-Ras dynamics, and can inform studies on the development of direct inhibitors towards the treatment of K-RasG12D-driven cancers.

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Characterization of the Family I inorganic pyrophosphatase fromPyrococcus horikoshiiOT3

Archaea ◽

10.1155/2005/591628 ◽

2005 ◽

Vol 1 (6) ◽

pp. 385-389 ◽

Cited By ~ 10

Author(s):

Sung-Jong Jeon ◽

Kazuhiko Ishikawa

Keyword(s):

Maximum Velocity ◽

Heat Stability ◽

Hydrolytic Activity ◽

Heat Inactivation ◽

Inorganic Pyrophosphatase ◽

Gene Encoding ◽

Sds Page ◽

The Family ◽

Hydrolysis Of

A gene encoding for a putative Family inorganic pyrophosphatase (PPase, EC 3.6.1.1) from the hyperthermophilic archaeonPyrococcus horikoshiiOT3 was cloned and the biochemical characteristics of the resulting recombinant protein were examined. The gene (Accession No. 1907) fromP. horikoshiishowed some identity with other Family I inorganic pyrophosphatases from archaea. The recombinant PPase fromP. horikoshii(PhPPase) has a molecular mass of 24.5 kDa, determined by SDS-PAGE. This enzyme specifically catalyzed the hydrolysis of pyrophosphate and was sensitive to NaF. The optimum temperature and pH for PPase activity were 70 °C and 7.5, respectively. The half-life of heat inactivation was about 50 min at 105 °C. The heat stability ofPhPPase was enhanced in the presence of Mg2+. A divalent cation was absolutely required for enzyme activity, Mg2+being most effective; Zn2+, Co2+and Mn2+efficiently supported hydrolytic activity in a narrow range of concentrations (0.05– 0.5 mM). The Kmfor pyrophosphate and Mg2+were 113 and 303 µM, respectively; and maximum velocity,Vmax, was estimated at 930 U mg–1.

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Structural and Functional Characterization of New SsoPox Variant Points to the Dimer Interface as a Driver for the Increase in Promiscuous Paraoxonase Activity

International Journal of Molecular Sciences ◽

10.3390/ijms21051683 ◽

2020 ◽

Vol 21 (5) ◽

pp. 1683 ◽

Cited By ~ 2

Author(s):

Yoko Suzumoto ◽

Orly Dym ◽

Giovanni N. Roviello ◽

Franz Worek ◽

Joel L. Sussman ◽

...

Keyword(s):

Functional Characterization ◽

Catalytic Efficiency ◽

Hydrolytic Activity ◽

Nerve Agents ◽

Process Step ◽

Homologous Genes ◽

New Variant ◽

Genes Encoding ◽

Hydrolysis Of

Increasing attention is more and more directed toward the thermostable Phosphotriesterase-Like-Lactonase (PLL) family of enzymes, for the efficient and reliable decontamination of toxic nerve agents. In the present study, the DNA Staggered Extension Process (StEP) technique was utilized to obtain new variants of PLL enzymes. Divergent homologous genes encoding PLL enzymes were utilized as templates for gene recombination and yielded a new variant of SsoPox from Saccharolobus solfataricus. The new mutant, V82L/C258L/I261F/W263A (4Mut) exhibited catalytic efficiency of 1.6 × 105 M−1 s−1 against paraoxon hydrolysis at 70°C, which is more than 3.5-fold and 42-fold improved in comparison with C258L/I261F/W263A (3Mut) and wild type SsoPox, respectively. 4Mut was also tested with chemical warfare nerve agents including tabun, sarin, soman, cyclosarin and VX. In particular, 4Mut showed about 10-fold enhancement in the hydrolysis of tabun and soman with respect to 3Mut. The crystal structure of 4Mut has been solved at the resolution of 2.8 Å. We propose that, reorganization of dimer conformation that led to increased central groove volume and dimer flexibility could be the major determinant for the improvement in hydrolytic activity in the 4Mut.

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