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1.
Science ; 373(6553)2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34437092

RESUMO

Systematic and extensive investigation of enzymes is needed to understand their extraordinary efficiency and meet current challenges in medicine and engineering. We present HT-MEK (High-Throughput Microfluidic Enzyme Kinetics), a microfluidic platform for high-throughput expression, purification, and characterization of more than 1500 enzyme variants per experiment. For 1036 mutants of the alkaline phosphatase PafA (phosphate-irrepressible alkaline phosphatase of Flavobacterium), we performed more than 670,000 reactions and determined more than 5000 kinetic and physical constants for multiple substrates and inhibitors. We uncovered extensive kinetic partitioning to a misfolded state and isolated catalytic effects, revealing spatially contiguous regions of residues linked to particular aspects of function. Regions included active-site proximal residues but extended to the enzyme surface, providing a map of underlying architecture not possible to derive from existing approaches. HT-MEK has applications that range from understanding molecular mechanisms to medicine, engineering, and design.


Assuntos
Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Fosfatase Alcalina/antagonistas & inibidores , Fosfatase Alcalina/química , Biocatálise , Domínio Catalítico , Flavobacterium/enzimologia , Hidrólise , Cinética , Microfluídica , Modelos Moleculares , Mutação , Oxigênio/metabolismo , Fosfatos/metabolismo , Conformação Proteica , Dobramento de Proteína , Termodinâmica
2.
Sci Rep ; 10(1): 13775, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792608

RESUMO

Chitin is one of the most abundant renewable organic materials found on earth. The chitin utilization locus in Flavobacterium johnsoniae, which encodes necessary proteins for complete enzymatic depolymerization of crystalline chitin, has recently been characterized but no detailed structural information on the enzymes was provided. Here we present protein structures of the F. johnsoniae chitobiase (FjGH20) and chitinase B (FjChiB). FjGH20 is a multi-domain enzyme with a helical domain not before observed in other chitobiases and a domain organization reminiscent of GH84 (ß-N-acetylglucosaminidase) family members. The structure of FjChiB reveals that the protein lacks loops and regions associated with exo-acting activity in other chitinases and instead has a more solvent accessible substrate binding cleft, which is consistent with its endo-chitinase activity. Additionally, small angle X-ray scattering data were collected for the internal 70 kDa region that connects the N- and C-terminal chitinase domains of the unique 158 kDa multi-domain chitinase A (FjChiA). The resulting model of the molecular envelope supports bioinformatic predictions of the region comprising six domains, each with similarities to either Fn3-like or Ig-like domains. Taken together, the results provide insights into chitin utilization by F. johnsoniae and reveal structural diversity in bacterial chitin metabolism.


Assuntos
Acetilglucosaminidase/metabolismo , Domínio Catalítico/genética , Quitina/metabolismo , Quitinases/metabolismo , Flavobacterium/enzimologia , Acetilglucosaminidase/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Quitinases/genética , Cristalografia por Raios X , Flavobacterium/genética , Flavobacterium/metabolismo , Modelos Moleculares
3.
Mar Drugs ; 18(8)2020 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-32722647

RESUMO

Alginate oligosaccharides produced by enzymatic degradation show versatile physiological functions and biological activities. In this study, a new alginate lyase encoding gene alyS02 from Flavobacterium sp. S02 was recombinantly expressed at a high level in Yarrowia lipolytica, with the highest extracellular activity in the supernatant reaching 36.8 ± 2.1 U/mL. AlyS02 was classified in the polysaccharide lyase (PL) family 7. The optimal reaction temperature and pH of this enzyme were 30 °C and 7.6, respectively, indicating that AlyS02 is a cold-adapted enzyme. Interestingly, AlyS02 contained more than 90% enzyme activity at 25 °C, higher than other cold-adapted enzymes. Moreover, AlyS02 is a bifunctional alginate lyase that degrades both polyG and polyM, producing di- and trisaccharides from alginate. These findings suggest that AlyS02 would be a potent tool for the industrial applications.


Assuntos
Alginatos/metabolismo , Proteínas de Bactérias/metabolismo , Flavobacterium/enzimologia , Polissacarídeo-Liases/metabolismo , Proteínas de Bactérias/genética , Estabilidade Enzimática , Flavobacterium/genética , Concentração de Íons de Hidrogênio , Cinética , Filogenia , Polissacarídeo-Liases/genética , Proteínas Recombinantes/metabolismo , Alga Marinha/microbiologia , Especificidade por Substrato , Temperatura
4.
J Sep Sci ; 43(15): 3036-3044, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32388896

RESUMO

Complete heparin digestion with heparin lyase I and II results in a mixture of hexasaccharides and tetrasaccharides with 3-O-sulfo group-containing glucosamine residues at their reducing ends. Because these tetrasaccharides are derived from antithrombin III-binding sites of heparin, we examined whether this method could be applied to estimate the anticoagulant activity of heparin. Therefore, this paper presents a new low molecular weight heparin sample preparation method-chemical depolymerization. Qualitative analysis of the studied compounds and a comparison of their composition are an important contribution to the structural analysis of low molecular weight heparins, which has not been fully conducted so far. Qualitative on-line liquid chromatography-mass spectrometric analysis of these resistant oligosaccharides is also described in this paper.


Assuntos
Glucosamina/metabolismo , Heparina Liase/metabolismo , Heparina/análise , Heparina/metabolismo , Oligossacarídeos/metabolismo , Cromatografia Líquida de Alta Pressão , Flavobacterium/enzimologia , Glucosamina/química , Heparina Liase/química , Peso Molecular , Oligossacarídeos/química , Controle de Qualidade , Espectrometria de Massas por Ionização por Electrospray
5.
Curr Pharm Biotechnol ; 21(13): 1304-1315, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31995003

RESUMO

OBJECTIVE: Alkaline Carboxymethyl Cellulase (CMCase) is an attractive enzyme for the textile, laundry, pulp, and paper industries; however, commercial preparations with sufficient activity at alkaline conditions are scarce. METHODS: High CMCase-producing bacterial isolate, SX9-4, was screened out from soil bacteria, which was identified as Flavobacterium sp. on the basis of 16S rDNA sequencing. RESULTS: The optimum pH and temperature for CMCase reaction were 8.0 and 55°C, respectively. Alkaline CMCase was stable over wide pH (3.0-10.6) and temperature (25-55°C) ranges. Enzyme activity was significantly inhibited by the bivalent cations Mn2+ and Cu2+, and was activated by Fe2+. To improve the alkaline CMCase production of SX9-4, fermentation parameters were selected through onefactor- at-a-time and further carried out by response surface methodologies based on a central composite design. CONCLUSION: High CMCase production (57.18 U/mL) was achieved under the optimal conditions: 10.53 g/L carboxymethylcellulose sodium, 7.74 g/L glucose, 13.71 g/L peptone, and 5.27 g/L ammonium oxalate.


Assuntos
Carboximetilcelulose Sódica/metabolismo , Fermentação , Flavobacterium/isolamento & purificação , Microbiologia Industrial/métodos , Microbiologia do Solo , Carboximetilcelulose Sódica/isolamento & purificação , Ativação Enzimática , Flavobacterium/enzimologia , Flavobacterium/genética , Concentração de Íons de Hidrogênio , RNA Ribossômico 16S , Temperatura
6.
FEBS J ; 287(6): 1195-1207, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31552702

RESUMO

Glycoside hydrolase family (GH) 31 contains a large variety of enzymes, but the major members are enzymes that act on relatively small oligosaccharides such as α-glucosidase. Here, we determined the crystal structure of Flavobacterium johnsoniae dextranase (FjDex31A), an enzyme from F. johnsoniae that hydrolyzes a polysaccharide, dextran. FjDex31A is composed of four domains: an N-terminal domain, a catalytic domain, a proximal C-terminal domain, and a distal C-terminal domain, as observed in typical GH31 enzymes. However, the architecture of active site residues in FjDex31A, other than subsite -1, is markedly different from that of other GH31 enzymes. The FjDex31A structure in complex with isomaltotriose shows that Gly273 and Tyr524, both of which interact with an α-glucose residue at subsite -2, as well as Trp376 and Leu308-cisGln309, are especially unique to FjDex31A. Site-directed mutagenesis of Gly273 and Tyr524 resulted in a decrease in the hydrolysis of polysaccharides dextran and pullulan, as well as that of the disaccharide isomaltose. These results suggest that, regardless of the length of sugar chains of the substrates, binding of FjDex31A to the substrates at subsite -2 is likely to be important for its activity. DATABASE: Structural data are available in the Protein Data Bank under the accession numbers 6JR6, 6JR7, and 6JR8.


Assuntos
Dextranase/química , Dextranase/metabolismo , Flavobacterium/enzimologia , Polissacarídeos/química , Polissacarídeos/metabolismo , Cristalografia por Raios X , Hidrólise , Modelos Moleculares , Relação Estrutura-Atividade , Especificidade por Substrato
7.
Life Sci ; 238: 116894, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31626789

RESUMO

AIMS: MicroRNAs (miRs) and their importance in development, normal physiology, and disease have become increasingly recognized. Our laboratory is interested in miR-29 and its effects on lung development. These studies set out to identify optimal conditions for the measurement of miR-29 in heparinized, biobanked samples and to compare isoform expression patterns. MATERIALS AND METHODS: The efficiency of three distinct heparinases were tested using reverse transcriptase polymerase chain reaction (RT-PCR): recombinant F. Heparinum heparinase I; recombinant P. heparinus heparinase II; recombinant P. heparinus heparinase III; and heparinase I (B. efferthii-derived). The effects of freeze/thaws, and the relative expression of different miR-29 isoforms were also assessed using RT-PCR. KEY FINDINGS: Our investigations determined that heparinase 1 (recombinant F. Heparinum) and 2 (recombinant P. heparinus) at 1 or 2 h incubation efficiently neutralized heparin activity and prevented interference with the PCR. Also, a single freeze/thaw did not affect the measurement of miR-29-3p but multiple freeze/thaw cycles decreased the measureable miR levels. Finally, the -3p strand was most abundantly expressed in all three isoforms in both human and mouse plasma. SIGNIFICANCE: Our findings illustrate that specific conditions need to be optimized for the particular miR and the type of sample being tested.


Assuntos
Bancos de Espécimes Biológicos/normas , Heparina/sangue , MicroRNAs/sangue , Animais , Estudos de Coortes , Flavobacterium/enzimologia , Heparina Liase/metabolismo , Humanos , Lactente , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase , Polissacarídeo-Liases/metabolismo , Proteínas Recombinantes/metabolismo
8.
Microbiol Res ; 223-225: 13-21, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31178046

RESUMO

Flavobacterium sp. AUG42 is a cellulase-producing bacterium isolated from the Antarctic oligochaete Grania sp. (Annelida). In this work, we report that AUG42 produces a glycoside hydrolase cocktail with CMCase, PASCase and cellobiase activities (optimum pHs and temperatures ranging from 5.5 to 6.5 and 40 to 50 °C, respectively). The time-course analyses of the bacterial growth and cellulase production showed that the cocktail has maximal activity at the stationary phase when growing at 16 °C with filter paper as a cellulosic carbon source, among the tested substrates. The analyses of the CAZome and the identification of secreted proteins by shotgun Mass Spectrometry analysis showed that five glycoside hydrolyses are present in the bacterial secretome, which probably cooperate in the degradation of the cellulosic substrates. Two of these glycoside hydrolyses may harbor putative carbohydrate binding modules, both with a cleft-like active site. The cellulolytic cocktail was assayed in saccharification experiments using carboxymethylcellulose as a substrate and results showed the release of glucose (a fermentable sugar) and other reducing-sugars, after 24 h incubation. The ecological relevance of producing cellulases in the Antarctic environment, as well as their potential use in the bio-refinery industry, are discussed.


Assuntos
Celulases/biossíntese , Celulases/química , Flavobacterium/enzimologia , Flavobacterium/metabolismo , Regiões Antárticas , Sequência de Bases , Carbono/metabolismo , Ciclo do Carbono , Carboximetilcelulose Sódica/metabolismo , Domínio Catalítico , Celulase , Celulases/genética , Celulose , Ensaios Enzimáticos , Fermentação , Flavobacterium/genética , Flavobacterium/crescimento & desenvolvimento , Glucose/metabolismo , Glicosídeo Hidrolases/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Especificidade por Substrato , Temperatura , beta-Glucosidase/metabolismo
9.
Biochemistry ; 58(14): 1845-1860, 2019 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-30855138

RESUMO

Class I ribonucleotide reductases (RNRs) share a common mechanism of nucleotide reduction in a catalytic α subunit. All RNRs initiate catalysis with a thiyl radical, generated in class I enzymes by a metallocofactor in a separate ß subunit. Class Id RNRs use a simple mechanism of cofactor activation involving oxidation of a MnII2 cluster by free superoxide to yield a metal-based MnIIIMnIV oxidant. This simple cofactor assembly pathway suggests that class Id RNRs may be representative of the evolutionary precursors to more complex class Ia-c enzymes. X-ray crystal structures of two class Id α proteins from Flavobacterium johnsoniae ( Fj) and Actinobacillus ureae ( Au) reveal that this subunit is distinctly small. The enzyme completely lacks common N-terminal ATP-cone allosteric motifs that regulate overall activity, a process that normally occurs by dATP-induced formation of inhibitory quaternary structures to prevent productive ß subunit association. Class Id RNR activity is insensitive to dATP in the Fj and Au enzymes evaluated here, as expected. However, the class Id α protein from Fj adopts higher-order structures, detected crystallographically and in solution. The Au enzyme does not exhibit these quaternary forms. Our study reveals structural similarity between bacterial class Id and eukaryotic class Ia α subunits in conservation of an internal auxiliary domain. Our findings with the Fj enzyme illustrate that nucleotide-independent higher-order quaternary structures can form in simple RNRs with truncated or missing allosteric motifs.


Assuntos
Domínio Catalítico , Desoxirribonucleotídeos/química , Conformação Proteica , Ribonucleotídeo Redutases/química , Actinobacillus/enzimologia , Actinobacillus/genética , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Regulação Alostérica , Sequência de Aminoácidos , Biocatálise , Cristalografia por Raios X , Desoxirribonucleotídeos/biossíntese , Desoxirribonucleotídeos/genética , Flavobacterium/enzimologia , Flavobacterium/genética , Modelos Moleculares , Filogenia , Ribonucleotídeo Redutases/classificação , Ribonucleotídeo Redutases/genética , Espalhamento a Baixo Ângulo , Homologia de Sequência de Aminoácidos , Difração de Raios X
10.
Appl Environ Microbiol ; 85(6)2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30635380

RESUMO

Hydrolytic extracellular enzymes degrading host tissues potentially play a role in bacterial pathogenesis. Flavobacterium psychrophilum is an important bacterial pathogen of salmonid fish reared in freshwater throughout the world. Diversity among isolates has been described at the phenotypic, serological, and genomic levels, but the links between these various traits remain poorly understood. Using a genome-wide association study, we identified a gene encoding a novel elastinolytic enzyme in F. psychrophilum To formally demonstrate enzymatic activity, this gene (FP0506 from strain JIP 02/86) was expressed in the elastinolysis-deficient strain OSU THCO2-90, resulting in proficient elastin-degrading cells. The encoded protein is predicted to be a cell-surface-exposed lipoprotein with no homology to previously reported elastases. FP0506 might belong to the zincin tribe and gluzincin clan of metalloproteases, and this new elastase-encoding gene seems to be present only in some members of the family Flavobacteriaceae IMPORTANCE Elastin is an important proteinaceous component of vertebrate connective tissues (e.g., blood vessels, lung, and skin), to which it confers elasticity. Elastases have been identified in a number of pathogenic bacteria. They are thought to be required for tissue penetration and dissemination, acting as "spreading factors." Flavobacterium psychrophilum is a devastating bacterial pathogen of salmonid fish (salmon and trout) that is responsible for severe economic losses worldwide. This pathogen displays strong proteolytic activities. Using a variety of techniques, including genome comparisons, we identified a gene encoding a novel elastase in F. psychrophilum The encoded protein is predicted to be a cell-surface-exposed lipoprotein with no homology to previously reported elastases. In addition, this elastase likely belongs to a new family of proteases that seems to be present only in some members of this important group of bacteria.


Assuntos
Proteínas de Bactérias/metabolismo , Elastina/metabolismo , Doenças dos Peixes/microbiologia , Infecções por Flavobacteriaceae/veterinária , Flavobacterium/enzimologia , Metaloproteases/metabolismo , Animais , Proteínas de Bactérias/genética , Infecções por Flavobacteriaceae/microbiologia , Flavobacterium/química , Flavobacterium/genética , Flavobacterium/isolamento & purificação , Genoma Bacteriano , Estudo de Associação Genômica Ampla , Metaloproteases/genética , Oncorhynchus mykiss/microbiologia
11.
Sci Rep ; 8(1): 16587, 2018 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-30410048

RESUMO

Iron(II)/α-ketoacid-dependent oxygenases (αKAOs) are enzymes that catalyze the oxidation of unactivated C-H bonds, mainly through hydroxylation. Among these, those that are active towards amino-acids and their derivatives are grouped in the Clavaminate Synthase Like (CSL) family. CSL enzymes exhibit high regio- and stereoselectivities with strict substrate specificity. This study reports the structural elucidation of two new regiodivergent members, KDO1 and KDO5, active towards lysine, and the structural and computational analysis of the whole family through modelling and classification of active sites. The structures of KDO1 and KDO5 in complex with their ligands show that one exact position in the active site controls the regioselectivity of the reaction. Our results suggest that the substrate specificity and high stereoselectivity typical of this family is linked to a lid that closes up in order to form a sub-pocket around the side chain of the substrate. This dynamic lid is found throughout the family with varying sequence and length and is associated with a conserved stable dimeric interface. Results from this study could be a starting-point for exploring the functional diversity of the CSL family and direct in vitro screening in the search for new enzymatic activities.


Assuntos
Actinobacteria/enzimologia , Flavobacterium/enzimologia , Oxigenases de Função Mista/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Oxigenases de Função Mista/metabolismo , Modelos Moleculares , Estrutura Terciária de Proteína , Especificidade por Substrato
12.
ACS Sens ; 3(12): 2675-2683, 2018 12 28.
Artigo em Inglês | MEDLINE | ID: mdl-30460848

RESUMO

Molecular self-replication is a fundamental function of all living organisms with the capability of templating and catalyzing its own synthesis, and it plays important roles in prebiotic chemical evolution and effective synthetic machineries. However, the construction of the self-replication system in vitro remains a great challenge and its application for biosensing is rare. Here, we demonstrate for the first time the construction of an in vitro enzymatic nucleic acid self-replication system and its application for amplified sensing of human 8-oxoguanine DNA glycosylase (hOGG1) based on autocatalytic self-replication-driven cascaded recycling amplification. In this strategy, hOGG1 excises 8-oxoguanine (8-oxoG) to unfold the hairpin substrate, activating the autonomous biocatalytic process with molecular beacons (MBs) as both the fuels for producing nucleic acid templates and the generators for signal output, leading to the continuous replication of biocatalytic nucleic acid templates and the repeated cleavage of MBs for an enhanced fluorescence signal. This strategy exhibits an extremely low detection limit of 4.3 × 10-7 U/µL and a large dynamic range of 5 orders of magnitude from 1 × 10-6 to 0.05 U/µL. Importantly, it can be applied for the detection of enzyme kinetic parameters, the screening of hOGG1 inhibitors, and the quantification of hOGG1 activity in even 1 single lung cancer cell, providing a new approach for biomedical research and clinical diagnosis.


Assuntos
DNA Glicosilases/análise , DNA/química , Ensaios Enzimáticos/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Células A549 , Técnicas Biossensoriais/métodos , Cloreto de Cádmio/química , DNA/genética , DNA Glicosilases/antagonistas & inibidores , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/química , Desoxirribonucleases de Sítio Específico do Tipo II/química , Inibidores Enzimáticos/química , Flavobacterium/enzimologia , Humanos , Limite de Detecção , Hibridização de Ácido Nucleico
13.
J Fish Dis ; 41(9): 1395-1402, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29893005

RESUMO

Flavobacterium columnare causes columnaris disease of farmed and wild freshwater fish. Skin mucus is an important factor in early stages of columnaris pathogenesis, albeit little studied. Our objectives were to (a) characterize the terminal glycosylation pattern (TGP) of catfish mucus, (b) determine the growth of F. columnare in formulated water (FW)-containing channel catfish (Ictalurus punctatus) or hybrid catfish (Ictalurus punctatus X Ictalurus furcatus) mucus and (c) examine extracellular protease activity of two F. columnare isolates differing in virulence. The TGP of catfish mucus by lectin binding was as follows: alpha-D-mannose/alpha-D-glucose >N-acetyl-beta-D-glucosamine >N-acetyl-beta-D-glucosamine/N-acetylneuraminic acid >N-acetyl-D-galactosamine >alpha-D-galactose/N-acetyl-alpha-D-galactosamine >beta-D-galactose = alpha-L-fucose. Virulence studies demonstrated isolate AL-02-36 was highly virulent in channel catfish fry (0.1 g) with cumulative mortality of 90%-100% versus 60% for isolate ALG-00-530 at equivalent doses (~3 × 106  CFU/ml); a similar result was observed in larger (0.7 g) catfish. In multiple experiments, F. columnare replicated (2-3 logs) and survived (28 days) in formulated water-containing catfish mucus. Highly virulent isolate AL-02-36 possessed at least 2.5- to fivefold higher protease activity following growth in mucus than the less virulent ALG-00-530. Flavobacterium columnare utilized catfish mucus as a nutrient source and mucus presence modulated extracellular protease production.


Assuntos
Peixes-Gato/microbiologia , Flavobacterium/enzimologia , Flavobacterium/crescimento & desenvolvimento , Muco/metabolismo , Peptídeo Hidrolases/metabolismo , Animais , Peixes-Gato/metabolismo , Meios de Cultura/química , Meios de Cultura/farmacologia , Doenças dos Peixes/microbiologia , Doenças dos Peixes/mortalidade , Infecções por Flavobacteriaceae/microbiologia , Infecções por Flavobacteriaceae/mortalidade , Flavobacterium/efeitos dos fármacos , Flavobacterium/patogenicidade , Galactose/metabolismo , Brânquias/microbiologia , Glicosilação , Lectinas/metabolismo , Muco/química , Peptídeo Hidrolases/biossíntese , Proteólise , Virulência
14.
Appl Microbiol Biotechnol ; 102(16): 6987-6996, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29948117

RESUMO

Carbohydrates are the product of carbon dioxide fixation by algae in the ocean. Their polysaccharides are depolymerized by marine bacteria, with a vast array of carbohydrate-active enzymes. These enzymes are important tools to establish biotechnological processes based on algal biomass. Green tides, which cover coastal areas with huge amounts of algae from the genus Ulva, represent a globally rising problem, but also an opportunity because their biomass could be used in biorefinery processes. One major component of their cell walls is the anionic polysaccharide ulvan for which the enzymatic depolymerization remains largely unknown. Ulvan lyases catalyze the initial depolymerization step of this polysaccharide, but only a few of these enzymes have been described. Here, we report the cloning, overexpression, purification, and detailed biochemical characterization of the endolytic ulvan lyase from Formosa agariphila KMM 3901T which is a member of the polysaccharide lyase family PL28. The identified biochemical parameters of the ulvan lyase reflect adaptation to the temperate ocean where the bacterium was isolated from a macroalgal surface. The NaCl concentration has a high influence on the turnover number of the enzyme and the affinity to ulvan. Divalent cations were shown to be essential for enzyme activity with Ca2+ likely being the native cofactor of the ulvan lyase. This study contributes to the understanding of ulvan lyases, which will be useful for future biorefinery applications of the abundant marine polysaccharide ulvan.


Assuntos
Flavobacterium/enzimologia , Polissacarídeo-Liases/metabolismo , Polissacarídeos/metabolismo , Flavobacterium/isolamento & purificação , Taiwan
15.
J Glob Antimicrob Resist ; 15: 55-60, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-29807204

RESUMO

OBJECTIVES: The aim of this study was to determine mutations associated with a quinolone-resistant (QR) phenotype of Flavobacterium columnare isolates. METHODS: The susceptibility of 53 F. columnare isolates to 11 antimicrobials, including 2 quinolones, was investigated by the disk diffusion method. Oxolinic acid (OXO) was subsequently chosen for minimum inhibitory concentration (MIC) assay. Sequence analysis of four genes within the quinolone resistance-determining regions (QRDRs) of OXO-resistant F. columnare compared with susceptible isolates was subsequently performed. RESULTS: The disk diffusion assay revealed that the majority of isolates were susceptible to all tested antimicrobials. However, 14 and 8 isolates were resistant to the quinolone antibiotics OXO and nalidixic acid, respectively. No multidrug resistance was observed. The MIC assay revealed five additional isolates that were resistant to OXO (≥4µg/mL), making a total of 19 OXO-resistant isolates observed in this study. DNA sequencing identified missense mutations both in parC and gyrA but not in gyrB or parE in QR F. columnare isolates. Mutation in parC resulted in the change His87→Tyr. For gyrA, 15 isolates of Thai origin exhibited a change at residue Ser83 to either Phe, Tyr or Ala, whereas 3 Vietnamese isolates contained two mutation sites (Ser83→Phe and Asp87→Tyr). CONCLUSION: This study is the first to reveal that QR phenotype F. columnare isolates harboured missense mutations both in parC and gyrA but not in gyrB or parE of the QRDRs.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/genética , DNA Girase/genética , DNA Topoisomerase IV/genética , Farmacorresistência Bacteriana , Infecções por Flavobacteriaceae/microbiologia , Flavobacterium/enzimologia , Mutação Puntual , Quinolonas/farmacologia , Proteínas de Bactérias/metabolismo , DNA Girase/metabolismo , DNA Topoisomerase IV/metabolismo , Flavobacterium/classificação , Flavobacterium/genética , Flavobacterium/isolamento & purificação , Humanos , Fenótipo
16.
Int J Biol Macromol ; 117: 62-71, 2018 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-29792968

RESUMO

Chitin and its derivatives are used for a variety of applications. Flavobacterium johnsoniae UW101 is an aerobic Gram-negative bacterium. Genome analysis of F. johnsoniae UW101 revealed the presence of 10 glycoside hydrolases (GHs) that may degrade or modify chitin. The gene encoding chitinase B (FjchiB), which encodes a single catalytic GH18 domain has been cloned and heterologously expressed in Escherichia coli. FjChiB was optimally active in 50 mM sodium citrate buffer (pH 6.0) at 40 °C. FjChiB was salt-tolerant and catalytically versatile, with substrate specificity towards 75% DDA (degree of de-acetylation) chitosan, followed by colloidal chitin. Chitotetraose (DP4) was the shortest of the oligomeric substrates used by FjChiB. The Km and Vmax values of FjChiB for colloidal chitin were 49.38 mg/ml and 11.2 nanokat mg-1, respectively. The overall catalytic efficiency (kcat/Km) of FjChiB was 1.40 × 103 mg-1 ml s-1. FjChiB exhibited transglycosylation (TG) with chitopentaose (DP5) and chitohexaose (DP6) substrates. The TG by FjChiB was fine-tuned by introducing a tryptophan (G106W) and asparagine (D148N) in the highly conserved catalytic groove and catalytic center, respectively. Hydrolytic products profile and homology modelling indicated that FjChiB is an endochitinase that holds promise for the conversion of chitin into useful products through both TG and/or hydrolysis.


Assuntos
Quitina/análogos & derivados , Quitinases/química , Quitinases/metabolismo , Flavobacterium/enzimologia , Quitina/biossíntese , Quitina/química , Quitinases/genética , Clonagem Molecular , Ativação Enzimática , Flavobacterium/genética , Expressão Gênica , Glicosilação , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Conformação Molecular , Mutagênese Sítio-Dirigida , Proteínas Recombinantes , Tolerância ao Sal , Especificidade por Substrato , Temperatura
17.
Int J Biol Macromol ; 116: 591-598, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29775704

RESUMO

The present work explores a rare cyanide dihydratase of Flavobacterium indicum MTCC 6936 for its potential of cyanide degradation. The enzyme is purified to 12 fold with a yield of 76%. SDS and native-PAGE analysis revealed that enzyme was monomer of 40 kDa size. The enzyme works well in mesophilic range at wide array of pH. The thermostability profile of cyanide dihydratase revealed that the enzyme is quite stable at 30 °C and 35 °C with half-life of 6 h 30 min and 5 h respectively. Km and Vmax for cyanide dihydratase of F. indicum was measured to be 4.76 mM and 45 U mg-1 with kcat calculated to be 27.3 s-1 and specificity constant (kcat/Km) to be around 5.67 mM-1 s-1. MALDI-TOF analysis of purified protein revealed that the amino acid sequence has 50% and 43% sequence identity with putative amino acid sequence of F. indicum and earlier reported cyanide dihydratase of Bacillus pumilus respectively. Homology modeling studies of cyanide dihydratase of F. indicum predicted the catalytic triad of the enzyme indicating Cys at 164, Glu at 46 and Lys at 130th position. The purified enzyme has potential applications in bioremediation and analytical sector.


Assuntos
Proteínas de Bactérias , Flavobacterium , Hidrolases , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Estabilidade Enzimática , Flavobacterium/enzimologia , Flavobacterium/crescimento & desenvolvimento , Hidrolases/biossíntese , Hidrolases/química , Hidrolases/isolamento & purificação
18.
Biochemistry ; 57(18): 2679-2693, 2018 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-29609464

RESUMO

A ribonucleotide reductase (RNR) from Flavobacterium johnsoniae ( Fj) differs fundamentally from known (subclass a-c) class I RNRs, warranting its assignment to a new subclass, Id. Its ß subunit shares with Ib counterparts the requirements for manganese(II) and superoxide (O2-) for activation, but it does not require the O2--supplying flavoprotein (NrdI) needed in Ib systems, instead scavenging the oxidant from solution. Although Fj ß has tyrosine at the appropriate sequence position (Tyr 104), this residue is not oxidized to a radical upon activation, as occurs in the Ia/b proteins. Rather, Fj ß directly deploys an oxidized dimanganese cofactor for radical initiation. In treatment with one-electron reductants, the cofactor can undergo cooperative three-electron reduction to the II/II state, in contrast to the quantitative univalent reduction to inactive "met" (III/III) forms seen with I(a-c) ßs. This tendency makes Fj ß unusually robust, as the II/II form can readily be reactivated. The structure of the protein rationalizes its distinctive traits. A distortion in a core helix of the ferritin-like architecture renders the active site unusually open, introduces a cavity near the cofactor, and positions a subclass-d-specific Lys residue to shepherd O2- to the Mn2II/II cluster. Relative to the positions of the radical tyrosines in the Ia/b proteins, the unreactive Tyr 104 of Fj ß is held away from the cofactor by a hydrogen bond with a subclass-d-specific Thr residue. Structural comparisons, considered with its uniquely simple mode of activation, suggest that the Id protein might most closely resemble the primordial RNR-ß.


Assuntos
Flavoproteínas/química , Manganês/química , Ribonucleotídeo Redutases/química , Superóxidos/química , Catálise , Domínio Catalítico , Flavobacterium/química , Flavobacterium/enzimologia , Flavoproteínas/metabolismo , Ferro/química , Oxirredução , Oxigênio/química , Ribonucleotídeo Redutases/classificação , Ribonucleotídeo Redutases/metabolismo , Tirosina/química
19.
Nature ; 557(7703): 123-126, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29695868

RESUMO

Alternative complex III (ACIII) is a key component of the respiratory and/or photosynthetic electron transport chains of many bacteria1-3. Like complex III (also known as the bc1 complex), ACIII catalyses the oxidation of membrane-bound quinol and the reduction of cytochrome c or an equivalent electron carrier. However, the two complexes have no structural similarity4-7. Although ACIII has eluded structural characterization, several of its subunits are known to be homologous to members of the complex iron-sulfur molybdoenzyme (CISM) superfamily 8 , including the proton pump polysulfide reductase9,10. We isolated the ACIII from Flavobacterium johnsoniae with native lipids using styrene maleic acid copolymer11-14, both as an independent enzyme and as a functional 1:1 supercomplex with an aa3-type cytochrome c oxidase (cyt aa3). We determined the structure of ACIII to 3.4 Å resolution by cryo-electron microscopy and constructed an atomic model for its six subunits. The structure, which contains a [3Fe-4S] cluster, a [4Fe-4S] cluster and six haem c units, shows that ACIII uses known elements from other electron transport complexes arranged in a previously unknown manner. Modelling of the cyt aa3 component of the supercomplex revealed that it is structurally modified to facilitate association with ACIII, illustrating the importance of the supercomplex in this electron transport chain. The structure also resolves two of the subunits of ACIII that are anchored to the lipid bilayer with N-terminal triacylated cysteine residues, an important post-translational modification found in numerous prokaryotic membrane proteins that has not previously been observed structurally in a lipid bilayer.


Assuntos
Microscopia Crioeletrônica , Grupo dos Citocromos c/química , Grupo dos Citocromos c/ultraestrutura , Citocromos a3/química , Citocromos a3/ultraestrutura , Citocromos a/química , Citocromos a/ultraestrutura , Complexo III da Cadeia de Transporte de Elétrons/química , Complexo III da Cadeia de Transporte de Elétrons/ultraestrutura , Flavobacterium/enzimologia , Cisteína/química , Cisteína/metabolismo , Grupo dos Citocromos c/metabolismo , Citocromos a/metabolismo , Citocromos a3/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Heme/análogos & derivados , Heme/química , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Lipídeos/química , Modelos Moleculares , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Oxirredução , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo
20.
Int J Biol Macromol ; 115: 176-184, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29578011

RESUMO

An enzyme aggregate of alginate lyase (EC 4.2.2.3) from flavobactierium was prepared using ammonium sulfate. The resultant aggregates upon cross-linking with glutaraldehyde produced insoluble and catalytically active cross-linked enzyme aggregate (CLEA) enzyme. The catalytic activity and stability of the cross-linked enzyme aggregate of alginate lyase (CLEA-AL) was studied in the presence of various pH, temperatures and organic solvents. Reusability, storage stability and surface morphology of the CLEA-AL were also studied. The native enzyme and CLEA-AL exhibited maximum enzyme activity at pH of 6.3 and at a temperature of 40°C. The CLEA-AL has good stability in nonpolar organic solvents and is thermally stable up to 50°C over a period of 8h. By encapsulating CLEA-AL into alginate hydrogel, we demonstrate that alginate hydrogels can be enzymatically degraded in a controlled fashion. The results also showed that degradation of alginate hydrogel with CLEA-AL incorporated beads is slower than native enzyme and therefore, CLEA-AL can be used for controlled degradation and release of various biologics from the degrading gel.


Assuntos
Alginatos/química , Engenharia , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Polissacarídeo-Liases/química , Polissacarídeo-Liases/metabolismo , Animais , Estabilidade Enzimática/efeitos dos fármacos , Flavobacterium/enzimologia , Ácido Glucurônico/química , Glutaral/química , Ácidos Hexurônicos/química , Solventes/farmacologia
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