scholarly journals Structural and biochemical analyses of selectivity determinants in chimeric Streptococcus Class A sortase enzymes

2021 ◽  
Author(s):  
Melody Gao ◽  
D. Alex Johnson ◽  
Isabel M. Piper ◽  
Hanna M. Kodama ◽  
Justin E. Svendsen ◽  
...  
Keyword(s):  
Class A ◽  
Biochemical Analyses

scholarly journals Nacubactam Enhances Meropenem Activity against Carbapenem-Resistant Klebsiella pneumoniae Producing KPC

2019 ◽  
Vol 63 (8) ◽  
Author(s):  
Melissa D. Barnes ◽  
Magdalena A. Taracila ◽  
Caryn E. Good ◽  
Saralee Bajaksouzian ◽  
Laura J. Rojas ◽  
...  
Keyword(s):  
Molecular Modeling ◽  
Klebsiella Pneumoniae ◽  
The United States ◽  
Alternative Mechanism ◽  
Enhancer Activity ◽  
Content Type ◽  
Class A ◽  
Carbapenem Resistant ◽  
Biochemical Analyses ◽  
Molecular Modeling And Docking

ABSTRACT Carbapenem-resistant Enterobacteriaceae (CRE) are resistant to most antibiotics, making CRE infections extremely difficult to treat with available agents. Klebsiella pneumoniae carbapenemases (KPC-2 and KPC-3) are predominant carbapenemases in CRE in the United States. Nacubactam is a bridged diazabicyclooctane (DBO) β-lactamase inhibitor that inactivates class A and C β-lactamases and exhibits intrinsic antibiotic and β-lactam “enhancer” activity against Enterobacteriaceae. In this study, we examined a collection of meropenem-resistant K. pneumoniae isolates carrying blaKPC-2 or blaKPC-3; meropenem-nacubactam restored susceptibility. Upon testing isogenic Escherichia coli strains producing KPC-2 variants with single-residue substitutions at important Ambler class A positions (K73, S130, R164, E166, N170, D179, K234, E276, etc.), the K234R variant increased the meropenem-nacubactam MIC compared to that for the strain producing KPC-2, without increasing the meropenem MIC. Correspondingly, nacubactam inhibited KPC-2 (apparent Ki [Ki app] = 31 ± 3 μM) more efficiently than the K234R variant (Ki app = 270 ± 27 μM) and displayed a faster acylation rate (k2/K), which was 5,815 ± 582 M−1 s−1 for KPC-2 versus 247 ± 25 M−1 s−1 for the K234R variant. Unlike avibactam, timed mass spectrometry revealed an intact sulfate on nacubactam and a novel peak (+337 Da) with the K234R variant. Molecular modeling of the K234R variant showed significant catalytic residue (i.e., S70, K73, and S130) rearrangements that likely interfere with nacubactam binding and acylation. Nacubactam’s aminoethoxy tail formed unproductive interactions with the K234R variant’s active site. Molecular modeling and docking observations were consistent with the results of biochemical analyses. Overall, the meropenem-nacubactam combination is effective against carbapenem-resistant K. pneumoniae. Moreover, our data suggest that β-lactamase inhibition by nacubactam proceeds through an alternative mechanism compared to that for avibactam.

scholarly journals Structural and biochemical analyses of selectivity determinants in chimeric Streptococcus Class A sortase enzymes

2021 ◽  
Author(s):  
Melody Gao ◽  
David Alex Johnson ◽  
Isabel M. Piper ◽  
Hanna M. Kodama ◽  
Justin E. Svendsen ◽  
...  
Keyword(s):  
Sequence Variation ◽  
Structural Model ◽  
Large Degree ◽  
Structural Determinants ◽  
Class A ◽  
C Terminus ◽  
Monomeric Structure ◽  
Biochemical Analyses ◽  
Related Proteins

Sequence variation in related proteins is an important characteristic that modulates activity and selectivity. An example of a protein family with a large degree of sequence variation is that of bacterial sortases, which are cysteine transpeptidases on the surface of gram positive bacteria. Class A sortases are responsible for attachment of diverse proteins to the cell wall to facilitate environmental adaption and interaction. These enzymes are also used in protein engineering applications for sortase-mediated ligations (SML) or sortagging of protein targets. We previously investigated SrtA from Streptococcus pneumoniae, identifying a number of putative β7-β8 loop mediated interactions that affected in vitro enzyme function. We identified residues that contributed to the ability of S. pneumoniae SrtA to recognize several amino acids at the P1' position of the substrate motif, underlined in LPXTG, in contrast to the strict P1' Gly recognition of SrtA from Staphylococcus aureus. However, motivated by the lack of a structural model for the active, monomeric form of S. pneumoniae SrtA, here, we expanded our studies to other Streptococcus SrtA proteins. We solved the first monomeric structure of S. agalactiae SrtA which includes the C-terminus, and three others of β7-β8 loop chimeras from S. pyogenes and S. agalactiae SrtA. These structures and accompanying biochemical data support our previously identified β7-β8 loop mediated interactions and provide additional insight into their role in Class A sortase substrate selectivity. A greater understanding of individual SrtA sequence and structural determinants of target selectivity can facilitate the design or discovery of improved sortagging tools.

scholarly journals CTX-M-Type Extended-Spectrum β-Lactamase That Hydrolyzes Ceftazidime through a Single Amino Acid Substitution in the Omega Loop

2001 ◽  
Vol 45 (12) ◽  
pp. 3355-3361 ◽  
Author(s):  
Laurent Poirel ◽  
Thierry Naas ◽  
Isabelle Le Thomas ◽  
Amal Karim ◽  
Edouard Bingen ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Substitution ◽  
Rectal Swab ◽  
Single Amino Acid ◽  
E Coli ◽  
Class A ◽  
Omega Loop ◽  
Similar Restriction ◽  
Biochemical Analyses ◽  
Hydrolysis Of

ABSTRACT Escherichia coli ILT-1, Klebsiella pneumoniae ILT-2, and K. pneumoniaeILT-3 were isolated in May 1999 in Paris, France, from a rectal swab of a hospitalized 5-month-old girl. These isolates had a clavulanic acid-inhibited substrate profile that included expanded-spectrum cephalosporins. The MICs of cefotaxime were higher for E. coli ILT-1 and K. pneumoniae ILT-2 than for K. pneumoniae ILT-3, while the opposite was found for the MICs of ceftazidime. Genetic and biochemical analyses revealed that E. coli ILT-1 and K. pneumoniae ILT-2 produced the CTX-M-18 β-lactamase, while K. pneumoniae ILT-3 produced the CTX-M-19 β-lactamase. The amino acid sequence of the CTX-M-18 β-lactamase differed from that of the CTX-M-9 β-lactamase by an Ala-to-Val change at position 231, while CTX-M-19 possessed an additional Pro-to-Ser change at position 167 in the omega loop of Ambler class A enzymes. The latter amino acid substitution may explain the CTX-M-19-mediated hydrolysis of ceftazidime, which has not been reported for other CTX-M-type enzymes. Thebla CTX-M-18 andbla CTX-M-19 genes were located on transferable plasmids that varied in size (ca. 60 and 50 kb, respectively) but that showed similar restriction patterns.

Visualization of the Platelet-Plasma Interface

1977 ◽  
Vol 35 ◽  
pp. 494-495
Author(s):  
D. C. Brindley ◽  
M. McGill
Keyword(s):  
Platelet Rich Plasma ◽  
Coagulation Factors ◽  
Platelet Membrane ◽  
Rabbit Platelet ◽  
Surface Coat ◽  
Special Relationship ◽  
Routine Method ◽  
Embedding Technique ◽  
Biochemical Analyses ◽  
Adsorptive Properties

Morphological and cytochemical studies of platelets have reported a surface coat, or glycocalyx, external to the plasma membrane (1). Biochemical analyses have likewise confirmed the highly adsorptive properties of platelets as transporters of coagulation factors (2). However, visualization of the platelet membrane by conventional EM procedures does not reflect this special relationship between the platelet and its plasma environment. By the routine method of alcohol-propylene oxide dehydration for Epon embedding, the lipid bilayer nature of the platelet membrane appears similar to other blood cells (Fig. 1). A new rapid embedding technique using dimethoxypropane (DMP) as dehydrating agent (13) has permitted ultrastructural analyses of the surface features of the platelet-plasma interface.Aliquots of human or rabbit platelet-rich plasma (PRP) were added to equal volumes of 6% glutaraldehyde in Millonig's buffer at 37° for 45 minutes, rinsed in buffer and postfixed in 1% osmium in Millonig's buffer for 45 minutes.

Morphological and biochemical analyses of changes in rat hepatocytes related to wine and ethanol consumption

1984 ◽  
Vol 42 ◽  
pp. 232-233
Author(s):  
S.M. Geyer ◽  
C.L. Mendenhall ◽  
J.T. Hung ◽  
E.L. Cardell ◽  
R.L. Drake ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Enzyme Activity ◽  
Malic Enzyme ◽  
Smooth Endoplasmic Reticulum ◽  
Rat Hepatocytes ◽  
Mature Male ◽  
Treatment Groups ◽  
Malic Enzyme Activity ◽  
Biochemical Analyses

Thirty-three mature male Holtzman rats were randomly placed in 3 treatment groups: Controls (C); Ethanolics (E); and Wine drinkers (W). The animals were fed synthetic diets (Lieber type) with ethanol or wine substituted isocalorically for carbohydrates in the diet of E and W groups, respectively. W received a volume of wine which provided the same gram quantity of alcohol consumed by E. The animals were sacrificed by decapitation after 6 weeks and the livers processed for quantitative triglycerides (T3), proteins, malic enzyme activity (MEA), light microscopy (LM) and electron microscopy (EM). Morphometric analysis of randomly selected LM and EM micrographs was performed to determine organellar changes in centrilobular (CV) and periportal (PV) regions of the liver. This analysis (Table 1) showed that hepatocytes from E were larger than those in C and W groups. Smooth endoplasmic reticulum decreased in E and increased in W compared to C values.

Important Compounds in the Cocaine Class: A Synthesis Overview

1991 ◽  
Author(s):  
F.I. Carroll ◽  
◽  
Anita H. Lewin
Keyword(s):  
Class A

DISTRIBUIÇÃO DA EVAPORAÇÃO EM ESTUFA PLÁSTICA NA PRIMAVERA

Irriga ◽  
2001 ◽  
Vol 6 (3) ◽  
pp. 120-127
Author(s):  
Reginaldo Ferreira Santos ◽  
Antonio Evaldo Klar
Keyword(s):  
Spatial Variability ◽  
Pan Evaporation ◽  
Spring Season ◽  
Class A ◽  
Agricultural Sciences ◽  
E Mail ◽  
Center Area ◽  
Class A Pan

DISTRIBUIÇÃO DA EVAPORAÇÃO EM ESTUFA PLÁSTICA NA PRIMAVERA  Reginaldo Ferreira SantosCentro de Ciências Exatas e Tecnológica da UNIOESTE- CP 711CEP 858114-110, Cascavel, PR - Fone: 0XX45 2203155.  E-mail: [email protected] Evaldo KlarDepartamento de Engenharia Rural - Faculdade de Ciências Agronômica- UNESP - CEP 18603-970 - Botucatu, SP. CP: 237.  E-mail:  [email protected]  1  RESUMO O presente trabalho teve como objetivo avaliar a distribuição da evaporação no interior de uma estufa plástica, com uma cultura de pimentão, através da variabilidade espacial e comparar a evaporação dos microevaporímetros com os valores do Tanque classe "A". O experimento foi conduzido no Campus da Universidade Estadual Paulista - FCA/UNESP, no período de primavera, em estufa plástica de polietileno de baixa densidade (PEBD). Na distribuição da evaporação em estufa com orientação norte/sul, verificou-se que as maiores evaporações ocorreram nas extremidades sul e norte tendente ao lado oeste. Já as menores evaporações localizaram-se no centro. No período de primavera, a evaporação média nos microevaporímetros superestimou em 55% a evaporação determinada no Tanque classe "A". UNITERMOS: evaporação, geoestatística, estufa.  SANTOS, R.F, KLAR, A.E.  EVAPORATION DISTRIBUTION INSIDE A PLASTIC TUNNEL IN THE SPRING SEASON  2  ABSTRACT                 The main aim of this study was to verify the evaporation distribution inside a plastic tunnel, with pepper crop, oriented to north/south, through spatial variability and to compare Class A Pan evaporation to punctual evaporations of 40 equidistant microevaporimeters placed from 50cm the soil. The study was carried out at the College of Agricultural Sciences/UNESP, Botucatu – SP in the spring season.  The highest evaporation occurred next to north and to south sides of the tunnel, with tendency to west. Consequently, the lowest evaporations occurred at the center area. The microevaporimeter evaporations were 55% higher than those obtained from Class A Pan. KEYWORDS: evaporation distribution, microevaporimeter.

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