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Frontiers in Bioengineering and... 2019Most of biochemical and mutagenesis studies performed with L-threonine aldolases were done with respect to natural activity, the cleavage of L-threonine and sometimes...
Most of biochemical and mutagenesis studies performed with L-threonine aldolases were done with respect to natural activity, the cleavage of L-threonine and sometimes L-β-phenylserine. However, the properties of variants and the impact of mutations on the product synthesis are more interesting from an applications point of view. Here we performed site-directed mutagenesis of active site residues of L-threonine aldolase from to analyze their impact on the retro-aldol activity and on the aldol synthesis of L-β-phenylserine and L-α-alkyl-β-phenylserines. Consequently, reduced retro-aldol activity upon mutation of catalytically important residues led to increased conversions and diastereoselectivities in the synthetic direction. Thus, L-β-phenylserine can be produced with conversions up to 60% and .'s up to 80% () under kinetic control. Furthermorem, the donor specificity of L-threonine aldolase was increased upon mutation of active site residues, which enlarged the pocket size for an efficient binding and stabilization of donor molecules in the active site. This study broadens the knowledge about L-threonine aldolase catalyzed reactions and improves the synthetic protocols for the biocatalytic asymmetric synthesis of unnatural amino acids.
PubMed: 31192202
DOI: 10.3389/fbioe.2019.00119 -
FEBS Open Bio Jun 2018Understanding the role of specific amino acid residues in the molecular mechanism of a protein's function is one of the most challenging problems in modern biology. A...
Understanding the role of specific amino acid residues in the molecular mechanism of a protein's function is one of the most challenging problems in modern biology. A systematic bioinformatic analysis of protein families and superfamilies can help in the study of structure-function relationships and in the design of improved variants of enzymes/proteins, but represents a methodological challenge. The pyridoxal-5'-phosphate (PLP)-dependent enzymes are catalytically diverse and include the aspartate aminotransferase superfamily which implements a common structural framework known as type fold I. In this work, the recently developed bioinformatic online methods Mustguseal and Zebra were used to collect and study a large representative set of the aspartate aminotransferase superfamily with high structural, but low sequence similarity to l-threonine aldolase from (LTAaj), in order to identify conserved positions that provide general properties in the superfamily, and to reveal family-specific positions (FSPs) responsible for functional diversity. The roles of the identified residues in the catalytic mechanism and reaction specificity of LTAaj were then studied by experimental site-directed mutagenesis and molecular modelling. It was shown that FSPs determine reaction specificity by coordinating the PLP cofactor in the enzyme's active centre, thus influencing its activation and the tautomeric equilibrium of the intermediates, which can be used as hotspots to modulate the protein's functional properties. Mutagenesis at the selected FSPs in LTAaj led to a reduction in a native catalytic activity and increased the rate of promiscuous reactions. The results provide insight into the structural basis of catalytic promiscuity of the PLP-dependent enzymes and demonstrate the potential of bioinformatic analysis in studying structure-function relationship in protein superfamilies.
PubMed: 29928580
DOI: 10.1002/2211-5463.12441 -
Genome Announcements Jun 2018We report here the whole-genome sequencing results of two bacterial isolates, IMET J and IMET F, that inhibit (possibly due to denitrifying gene clusters) and promote...
Draft Genome Sequences of Cloacibacterium normanense IMET F, a Microalgal Growth-Promoting Bacterium, and Aeromonas jandaei IMET J, a Microalgal Growth-Inhibiting Bacterium.
We report here the whole-genome sequencing results of two bacterial isolates, IMET J and IMET F, that inhibit (possibly due to denitrifying gene clusters) and promote (possibly due to an ammonification system), respectively, the growth of the microalgal strains HTB1 and 1807.
PubMed: 29903817
DOI: 10.1128/genomeA.00503-18 -
Microbes and Environments Sep 2016A facultative anaerobic bacterium, designated as strain SCS5, was isolated from the anodic biofilm of a mediator-less microbial fuel cell using acetate as the electron...
Electrochemical Characterization of a Novel Exoelectrogenic Bacterium Strain SCS5, Isolated from a Mediator-Less Microbial Fuel Cell and Phylogenetically Related to Aeromonas jandaei.
A facultative anaerobic bacterium, designated as strain SCS5, was isolated from the anodic biofilm of a mediator-less microbial fuel cell using acetate as the electron donor and α-FeOOH as the electron acceptor. The isolate was Gram-negative, motile, and shaped as short rods (0.9-1.3 μm in length and 0.4-0.5 μm in width). A phylogenetic analysis of the 16S rRNA, gyrB, and rpoD genes suggested that strain SCS5 belonged to the Aeromonas genus in the Aeromonadaceae family and exhibited the highest 16S rRNA gene sequence similarity (99.45%) with Aeromonas jandaei ATCC 49568. However, phenotypic, cellular fatty acid profile, and DNA G+C content analyses revealed that there were some distinctions between strain SCS5 and the type strain A. jandaei ATCC 49568. The optimum growth temperature, pH, and NaCl (%) for strain SCS5 were 35°C, 7.0, and 0.5% respectively. The DNA G+C content of strain SCS5 was 59.18%. The isolate SCS5 was capable of reducing insoluble iron oxide (α-FeOOH) and transferring electrons to extracellular material (the carbon electrode). The electrochemical activity of strain SCS5 was corroborated by cyclic voltammetry and a Raman spectroscopic analysis. The cyclic voltammogram of strain SCS5 revealed two pairs of oxidation-reduction peaks under anaerobic and aerobic conditions. In contrast, no redox pair was observed for A. jandaei ATCC 49568. Thus, isolated strain SCS5 is a novel exoelectrogenic bacterium phylogenetically related to A. jandaei, but shows distinct electrochemical activity from its close relative A. jandaei ATCC 49568.
Topics: Aerobiosis; Aeromonas; Anaerobiosis; Bacterial Typing Techniques; Base Composition; Bioelectric Energy Sources; Cluster Analysis; Cytosol; DNA Gyrase; DNA, Bacterial; DNA, Ribosomal; DNA-Directed RNA Polymerases; Electrochemical Techniques; Electron Transport; Fatty Acids; Ferric Compounds; Hydrogen-Ion Concentration; Locomotion; Microscopy, Electron; Oxidation-Reduction; Phylogeny; RNA, Ribosomal, 16S; Sequence Analysis, DNA; Sigma Factor; Sodium Chloride; Temperature
PubMed: 27396922
DOI: 10.1264/jsme2.ME15185 -
Brazilian Journal of Microbiology :... Jul 2010It is known that Aeromonas spp. possess different chromosomal β-lactamase genes. Presence and phenotypic expression of bla TEM, bla SHV, and bla CTX-M ESBL-encoding...
It is known that Aeromonas spp. possess different chromosomal β-lactamase genes. Presence and phenotypic expression of bla TEM, bla SHV, and bla CTX-M ESBL-encoding genes were investigated in environmental water isolates of Aeromonas hydrophila and Aeromonas jandaei. Presence of bla SHV and bla CTX-M genes was not observed, and bla TEM gene was verified in 91% of the isolates. Sequencing of 10 fragments showed the occurrence of bla TEM-116.
PubMed: 24031548
DOI: 10.1590/S1517-83822010000300023 -
Revista Da Sociedade Brasileira de... 2008Aeromonas spp is recognized as pathogenic to humans after consumption of contaminated water and food. In the present investigation, 2,323 rectal swab samples from...
Aeromonas spp is recognized as pathogenic to humans after consumption of contaminated water and food. In the present investigation, 2,323 rectal swab samples from newborns hospitalized in Rio de Janeiro were evaluated with a view to isolating Aeromonas. The samples were collected and sent to the national reference laboratory for cholera and other bacterial intestinal infections, at the Oswaldo Cruz Institute of the Oswaldo Cruz Foundation. The swabs were subjected to enrichment in alkaline peptonated water with the addition of 1% sodium chloride (NaCl) and alkaline peptonated water plus 3% NaCl (37 degrees C/18-24h) and were streaked onto agar that was selective for Pseudomonas-Aeromonas (GSP Agar). Fifty-six Aeromonas strains were isolated, distributed as follows: Aeromonas caviae (42.8%), Aeromonas media (25%), Aeromonas veronii biogroup sobria (10.7%), Aeromonas hydrophila (9%), Aeromonas veronii biogroup veronii (5.3%), Aeromonas sobria (1.8%), Aeromonas jandaei (1.8%), Aeromonas schubertii (1.8%) and Aeromonas sp (1.8%). Resistance to one or more antimicrobial drugs was observed in 26.8% of the strains. Considering the importance of Aeromonas, there is an urgent need to warn about this in relation to nosocomial infection control.
Topics: Aeromonas; Anti-Bacterial Agents; Drug Resistance, Microbial; Humans; Infant, Newborn; Microbial Sensitivity Tests; Rectum
PubMed: 18545840
DOI: 10.1590/s0037-86822008000200009 -
Revista Chilena de Infectologia :... Jun 2007Extraintestinal infections caused by the genera Aeromonas, Vibrio and Plesiomonas have high morbidity and mortality rates in different areas of world. From January 2002...
Extraintestinal infections caused by the genera Aeromonas, Vibrio and Plesiomonas have high morbidity and mortality rates in different areas of world. From January 2002 to December 2003, the National Reference Laboratory for Acute Diarrhoeal Diseases of the Pedro Kourí Tropical Medicine Institute received 95 gram-negative, facultative anaerobic, oxidase positive bacilli strains from different extraintestinal specimen (blood, ear exudates, infected wounds, conjunctive exudates, urine, and catheters, among others) sent by different provincial laboratories along the country. Aeromonas caviae, Aeromonas veronii bv sobria, Aeromonas jandaei, Vibrio cholerae no-O1, Vibrio vulnificus, Vibrio fluvialis and Plesiomonas shigelloides were the species most frequently found in the sample analysed.
Topics: Aeromonas; Bacterial Typing Techniques; Cuba; Humans; Plesiomonas; Vibrio cholerae
PubMed: 17554439
DOI: 10.4067/s0716-10182007000300005 -
Applied and Environmental Microbiology Jan 2007The gut bacteria of the North American medicinal leech, Macrobdella decora, were characterized. Biochemical tests and DNA sequences indicated that Aeromonas jandaei is...
The gut bacteria of the North American medicinal leech, Macrobdella decora, were characterized. Biochemical tests and DNA sequences indicated that Aeromonas jandaei is the dominant culturable symbiont in leeches from a broad geographic area. In this work we identified a new habitat for A. jandaei, and here we suggest that there is unexpected specificity between leeches and Aeromonas species.
Topics: Aeromonas; Animals; Bacterial Typing Techniques; Gastrointestinal Tract; Leeches; North America; Phenotype; Phylogeny; Sequence Analysis, DNA; Symbiosis
PubMed: 17114316
DOI: 10.1128/AEM.01282-06 -
Applied and Environmental Microbiology Oct 2006Eighty-one tetracycline-resistant Aeromonas sp. strains were isolated from farm-raised catfish. Morphological and biochemical characteristics indicated that 23 of the 81...
Eighty-one tetracycline-resistant Aeromonas sp. strains were isolated from farm-raised catfish. Morphological and biochemical characteristics indicated that 23 of the 81 aeromonads were Aeromonas hydrophila, 7 isolates were Aeromonas trota, 6 isolates were Aeromonas caviae, 42 isolates were Aeromonas veronii, and 3 isolates were Aeromonas jandaei. However, the AluI and MboI restriction fragment length polymorphism (RFLP) patterns of the PCR-amplified 1.4-kb 16S rRNA gene from all 81 tetracycline-resistant aeromonads from catfish were identical to the RFLP banding patterns of A. veronii ATCC 35626, indicating that all 81 isolates were strains of A. veronii. A multiplex PCR assay successfully amplified the 5 tetracycline-resistant genes (tetA to E) from the genomic DNA of all 81 isolates. The assay determined that tetE was the dominant gene occurring in 73/81 (90.0%) of the aeromonads. Plasmids (2.0 to 20 kb) were isolated from 33 of the 81 isolates. Dendrogram analysis of the SpeI pulsed-field gel electrophoresis identified 15 distinct macrorestriction patterns among the isolates. Our results indicate the need for use of 16S rRNA in the identification of Aeromonas spp. and the prevalence of catfish as a reservoir of tet genes.
Topics: Aeromonas; Animals; Bacterial Typing Techniques; Catfishes; Drug Resistance, Bacterial; Electrophoresis, Gel, Pulsed-Field; Food Microbiology; Plasmids; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; RNA, Ribosomal, 16S; Tetracyclines
PubMed: 17021193
DOI: 10.1128/AEM.00271-06 -
Journal of Food Protection May 2001Numbers and species of motile Aeromonas were determined in freshly caught freshwater fish, in the surrounding environment, and also during iced chilled storage of fish... (Comparative Study)
Comparative Study
Numbers and species of motile Aeromonas were determined in freshly caught freshwater fish, in the surrounding environment, and also during iced chilled storage of fish specimens. Although no significant differences were observed in water samples, initial levels for skin, gill, and intestines were significantly lower in farmed rainbow trout (Oncorhynchus mykiss) than in wild brown trout (Salmo trutta) and pike (Esox lucius). During storage of wild specimens, naturally occurring aeromonads grew fairly well on the surfaces of skin and body cavity. Of 171 strains assigned to the genus Aeromonas, 88% were identified to phenospecies and putative genospecies level by using comprehensive biochemical schemes. The isolates were allocated to putative hybridization groups (HGs) 1 and 3 Aeromonas hydrophila (29%); putative HG 8 Aeromonas veronii biovar sobria (19%); putative HG 2 Aeromonas bestiarum (18%); putative HG 9 Aeromonas jandaei (16%); putative HGs 4 and 5a Aeromonas caviae (2%); putative HG 12 Aeromonas schubertii (2%); and putative HG 11 (unnamed, 0.6%). The remaining 20 isolates (12%) resembled A. schubertii but could not be allocated to currently recognized phenospecies or to putative HGs. Although cultured rainbow trout yielded strains of putative HGs 1, 4, and 8, which appear to be of major clinical importance, most isolates assigned to putative HGs 1 and 8 were recovered from pike. Differences among HGs found in wild animals could be related to their origin (unpolluted rivers for brown trout and urban rivers for pike). The recovery of these aeromonads species was not related to sampling site. The initial levels of motile aeromonads, their behavior during storage, and the strong potential spoilage activity of most isolates confirm that these bacteria can contribute to deterioration of iced wild freshwater fish. Although adequate cooking would inactivate motile aeromonads, the high incidence of isolates belonging to gastroenteritis-associated HGs should be regarded as a potential health concern, particularly for susceptible populations when there is a possibility of cross-contamination.
Topics: Aeromonas; Animals; Animals, Wild; Aquaculture; Esocidae; Fishes; Trout; Water Microbiology
PubMed: 11348001
DOI: 10.4315/0362-028x-64.5.687