Genetic and Biochemical Characterization of OXA-535, a Distantly Related OXA-48-Like β-Lactamase

ABSTRACT OXA-535 is a chromosome-encoded carbapenemase of Shewanella bicestrii JAB-1 that shares only 91.3% amino acid sequence identity with OXA-48. Catalytic efficiencies are similar to those of OXA-48 for most β-lactams, except for ertapenem, where a 2,000-fold-higher efficiency was observed with OXA-535. OXA-535 and OXA-436, a plasmid-encoded variant of OXA-535 differing by three amino acids, form a novel cluster of distantly related OXA-48-like carbapenemases. Comparison of blaOXA-535 and blaOXA-436 genetic environments suggests that an ISCR1 may be responsible for blaOXA-436 gene mobilization from the chromosome of Shewanella spp. to plasmids.

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GS32, a Novel Golgi SNARE of 32 kDa, Interacts Preferentially with Syntaxin 6

Molecular Biology of the Cell ◽

10.1091/mbc.10.1.119 ◽

1999 ◽

Vol 10 (1) ◽

pp. 119-134 ◽

Cited By ~ 54

Author(s):

Siew Heng Wong ◽

Yue Xu ◽

Tao Zhang ◽

Gareth Griffiths ◽

Stephen Loucian Lowe ◽

...

Keyword(s):

Amino Acid ◽

Golgi Apparatus ◽

Amino Acid Sequence ◽

Northern Blot Analysis ◽

Amino Acid Sequence Identity ◽

Biochemical Characterization ◽

Polyclonal Antibodies ◽

Sequence Identity ◽

Membrane Bound

Syntaxin 1, synaptobrevins or vesicle-associated membrane proteins, and the synaptosome-associated protein of 25 kDa (SNAP-25) are key molecules involved in the docking and fusion of synaptic vesicles with the presynaptic membrane. We report here the molecular, cell biological, and biochemical characterization of a 32-kDa protein homologous to both SNAP-25 (20% amino acid sequence identity) and the recently identified SNAP-23 (19% amino acid sequence identity). Northern blot analysis shows that the mRNA for this protein is widely expressed. Polyclonal antibodies against this protein detect a 32-kDa protein present in both cytosol and membrane fractions. The membrane-bound form of this protein is revealed to be primarily localized to the Golgi apparatus by indirect immunofluorescence microscopy, a finding that is further established by electron microscopy immunogold labeling showing that this protein is present in tubular-vesicular structures of the Golgi apparatus. Biochemical characterizations establish that this protein behaves like a SNAP receptor and is thus named Golgi SNARE of 32 kDa (GS32). GS32 in the Golgi extract is preferentially retained by the immobilized GST–syntaxin 6 fusion protein. The coimmunoprecipitation of syntaxin 6 but not syntaxin 5 or GS28 from the Golgi extract by antibodies against GS32 further sustains the preferential interaction of GS32 with Golgi syntaxin 6.

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Cloning, Expression, and Characterization of a New β-Agarase fromVibriosp. Strain CN41

Applied and Environmental Microbiology ◽

10.1128/aem.05364-11 ◽

2011 ◽

Vol 77 (19) ◽

pp. 7077-7079 ◽

Cited By ~ 32

Author(s):

Li Liao ◽

Xue-Wei Xu ◽

Xia-Wei Jiang ◽

Yi Cao ◽

Na Yi ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Amino Acid Sequence ◽

Glycoside Hydrolase ◽

Amino Acid Sequence Identity ◽

Sequence Identity

ABSTRACTA new agarase, AgaACN41, cloned fromVibriosp. strain CN41, consists of 990 amino acids, with only 49% amino acid sequence identity with known β-agarases. AgaACN41belongs to the GH50 (glycoside hydrolase 50) family but yields neoagarotetraose as the end product. AgaACN41was expressed and characterized.

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Biochemical characterization of a glucoamylase from Saccharomycopsis fibuligera R64

Biologia ◽

10.2478/s11756-010-0151-2 ◽

2011 ◽

Vol 66 (1) ◽

Cited By ~ 3

Author(s):

Dessy Natalia ◽

Keni Vidilaseris ◽

Pasjan Satrimafitrah ◽

Wangsa Ismaya ◽

Purkan ◽

...

Keyword(s):

Mass Spectrometry ◽

Amino Acid ◽

Amino Acid Sequence Identity ◽

Biochemical Characterization ◽

Saccharomycopsis Fibuligera ◽

Sequence Identity ◽

Ic50 Value ◽

High Amino Acid ◽

Ph Range

AbstractGlucoamylase from the yeast Saccharomycopsis fibuligera R64 (GLL1) has successfully been purified and characterized. The molecular mass of the enzyme was 56,583 Da as determined by mass spectrometry. The purified enzyme demonstrated optimum activity in the pH range of 5.6–6.4 and at 50°C. The activity of the enzyme was inhibited by acarbose with the IC50 value of 5 μM. GLL1 shares high amino acid sequence identity with GLU1 and GLA1, which are Saccharomycopsis fibuligera glucoamylases from the strains HUT7212 and KZ, respectively. The properties of GLL1, however, resemble that of GLU1. The elucidation of the primary structure of GLL1 contributes to the explanation of this finding.

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Biochemical characterization of a second family of human Ia molecules, HLA-DS, equivalent to murine I-A subregion molecules.

Journal of Experimental Medicine ◽

10.1084/jem.156.2.550 ◽

1982 ◽

Vol 156 (2) ◽

pp. 550-566 ◽

Cited By ~ 145

Author(s):

S M Goyert ◽

J E Shively ◽

J Silver

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Amino Acid Sequence Identity ◽

Biochemical Characterization ◽

Amino Acid Sequence Homology ◽

Molecular Weights ◽

Hla Dr ◽

D Region ◽

Polypeptide Chains

In mice, two families of structurally distinct Ia molecules, one designated I-A and the other I-E, have been identified and characterized. The HLA-DR molecules represent one family of human Ia molecules equivalent to the murine I-E molecules on the basis of amino acid sequence homology. We describe the isolation and biochemical characterization of a second family of human Ia molecules, designated HLA-DS for second D-region locus, equivalent to the murine I-A molecules. The human HLA-DS molecules consist of two polypeptide chains, DS alpha (37,000 mol wt) and DS beta (29,000 mol wt), with 73% amino acid sequence identity to the murine I-A molecules. Furthermore, the HLA-DS molecules are closely linked genetically to HLA-DR molecules, a situation analogous to that observed in mice. The similarity in molecular weights of the DR and DS molecules might explain why others have failed to identify the latter in man.

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Comparative Biochemical and Structural Analysis of Novel Cellulose Binding Proteins (Tāpirins) from Extremely Thermophilic Caldicellulosiruptor Species

Applied and Environmental Microbiology ◽

10.1128/aem.01983-18 ◽

2018 ◽

Vol 85 (3) ◽

Cited By ~ 7

Author(s):

Laura L. Lee ◽

William S. Hart ◽

Vladimir V. Lunin ◽

Markus Alahuhta ◽

Yannick J. Bomble ◽

...

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Microcrystalline Cellulose ◽

Binding Proteins ◽

Amino Acid Sequence Identity ◽

Plant Biomass ◽

Three Dimensional ◽

Content Type ◽

Sequence Identity ◽

Cellulose Binding

ABSTRACT Genomes of extremely thermophilic Caldicellulosiruptor species encode novel cellulose binding proteins, called tāpirins, located proximate to the type IV pilus locus. The C-terminal domain of Caldicellulosiruptor kronotskyensis tāpirin 0844 (Calkro_0844) is structurally unique and has a cellulose binding affinity akin to that seen with family 3 carbohydrate binding modules (CBM3s). Here, full-length and C-terminal versions of tāpirins from Caldicellulosiruptor bescii (Athe_1870), Caldicellulosiruptor hydrothermalis (Calhy_0908), Caldicellulosiruptor kristjanssonii (Calkr_0826), and Caldicellulosiruptor naganoensis (NA10_0869) were produced recombinantly in Escherichia coli and compared to Calkro_0844. All five tāpirins bound to microcrystalline cellulose, switchgrass, poplar, and filter paper but not to xylan. Densitometry analysis of bound protein fractions visualized by SDS-PAGE revealed that Calhy_0908 and Calkr_0826 (from weakly cellulolytic species) associated with the cellulose substrates to a greater extent than Athe_1870, Calkro_0844, and NA10_0869 (from strongly cellulolytic species). Perhaps this relates to their specific needs to capture glucans released from lignocellulose by cellulases produced in Caldicellulosiruptor communities. Calkro_0844 and NA10_0869 share a higher degree of amino acid sequence identity (>80% identity) with each other than either does with Athe_1870 (∼50%). The levels of amino acid sequence identity of Calhy_0908 and Calkr_0826 to Calkro_0844 were only 16% and 36%, respectively, although the three-dimensional structures of their C-terminal binding regions were closely related. Unlike the parent strain, C. bescii mutants lacking the tāpirin genes did not bind to cellulose following short-term incubation, suggesting a role in cell association with plant biomass. Given the scarcity of carbohydrates in neutral terrestrial hot springs, tāpirins likely help scavenge carbohydrates from lignocellulose to support growth and survival of Caldicellulosiruptor species. IMPORTANCE The mechanisms by which microorganisms attach to and degrade lignocellulose are important to understand if effective approaches for conversion of plant biomass into fuels and chemicals are to be developed. Caldicellulosiruptor species grow on carbohydrates from lignocellulose at elevated temperatures and have biotechnological significance for that reason. Novel cellulose binding proteins, called tāpirins, are involved in the way that Caldicellulosiruptor species interact with microcrystalline cellulose, and additional information about the diversity of these proteins across the genus, including binding affinity and three-dimensional structural comparisons, is provided here.

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Molecular Mechanism of Nicotine Degradation by a Newly Isolated Strain, Ochrobactrum sp. Strain SJY1

Applied and Environmental Microbiology ◽

10.1128/aem.02265-14 ◽

2014 ◽

Vol 81 (1) ◽

pp. 272-281 ◽

Cited By ~ 52

Author(s):

Hao Yu ◽

Hongzhi Tang ◽

Xiongyu Zhu ◽

Yangyang Li ◽

Ping Xu

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Amino Acid Sequence Identity ◽

Specific Activity ◽

Amine Oxidase ◽

Sole Source ◽

Content Type ◽

Sequence Identity ◽

Nicotine Degradation ◽

Functional Pathway

ABSTRACTA newly isolated strain, SJY1, identified asOchrobactrumsp., utilizes nicotine as a sole source of carbon, nitrogen, and energy. Strain SJY1 could efficiently degrade nicotine via a variant of the pyridine and pyrrolidine pathways (the VPP pathway), which highlights bacterial metabolic diversity in relation to nicotine degradation. A 97-kbp DNA fragment containing six nicotine degradation-related genes was obtained by gap closing from the genome sequence of strain SJY1. Three genes, designatedvppB,vppD, andvppE, in the VPP pathway were cloned and heterologously expressed, and the related proteins were characterized. ThevppBgene encodes a flavin-containing amine oxidase converting 6-hydroxynicotine to 6-hydroxy-N-methylmyosmine. Although VppB specifically catalyzes the dehydrogenation of 6-hydroxynicotine rather than nicotine, it shares higher amino acid sequence identity with nicotine oxidase (38%) from the pyrrolidine pathway than with its isoenzyme (6-hydroxy-l-nicotine oxidase, 24%) from the pyridine pathway. ThevppDgene encodes an NADH-dependent flavin-containing monooxygenase, which catalyzes the hydroxylation of 6-hydroxy-3-succinoylpyridine to 2,5-dihydroxypyridine. VppD shows 62% amino acid sequence identity with the hydroxylase (HspB) fromPseudomonas putidastrain S16, whereas the specific activity of VppD is ∼10-fold higher than that of HspB. VppE is responsible for the transformation of 2,5-dihydroxypyridine. Sequence alignment and phylogenetic analysis suggested that the VPP pathway, which evolved independently from nicotinic acid degradation, might have a closer relationship with the pyrrolidine pathway. The proteins and functional pathway identified here provide a sound basis for future studies aimed at a better understanding of molecular principles of nicotine degradation.

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The Class A β-Lactamase FTU-1 Is Native to Francisella tularensis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.05305-11 ◽

2011 ◽

Vol 56 (2) ◽

pp. 666-671 ◽

Cited By ~ 24

Author(s):

Nuno T. Antunes ◽

Hilary Frase ◽

Marta Toth ◽

Sergei B. Vakulenko

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Francisella Tularensis ◽

Amino Acid Sequence Identity ◽

Cysteine Residues ◽

Content Type ◽

Sequence Identity ◽

Class A

ABSTRACTThe class A β-lactamase FTU-1 produces resistance to penicillins and ceftazidime but not to any other β-lactam antibiotics tested. FTU-1 hydrolyzes penicillin antibiotics with catalytic efficiencies of 105to 106M−1s−1and cephalosporins and carbapenems with catalytic efficiencies of 102to 103M−1s−1, but the monobactam aztreonam and the cephamycin cefoxitin are not substrates for the enzyme. FTU-1 shares 21 to 34% amino acid sequence identity with other class A β-lactamases and harbors two cysteine residues conserved in all class A carbapenemases. FTU-1 is the first weak class A carbapenemase that is native toFrancisella tularensis.

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Nucleotide Sequence and Characterization of a Novel Cefotaxime-Hydrolyzing β-Lactamase (CTX-M-10) Isolated in Spain

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.45.2.616-620.2001 ◽

2001 ◽

Vol 45 (2) ◽

pp. 616-620 ◽

Cited By ~ 70

Author(s):

Antonio Oliver ◽

José Claudio Pérez-Dı́az ◽

Teresa M. Coque ◽

Fernando Baquero ◽

Rafael Cantón

Keyword(s):

Amino Acid ◽

Nucleotide Sequence ◽

Amino Acid Sequence Identity ◽

Penicillin G ◽

Nucleotide Substitutions ◽

Sequence Identity ◽

M 10 ◽

Hydrolysis Rates ◽

Kluyvera Ascorbata

ABSTRACT A cefotaxime-resistant, ceftazidime-susceptible Escherichia coli isolate was obtained from a patient with sepsis in 1997, from which a β-lactamase with a pI of 8.1 was cloned. Cephaloridine and cefotaxime relative hydrolysis rates were 167 and 81, respectively (penicillin G rate = 100), whereas ceftazidime hydrolysis was not detected. The nucleotide sequence revealed a bla gene related to that coding for CTX-M-3. Despite 21 nucleotide substitutions, only 2 determined amino acid changes (Ala27Val and Arg38Gln). The amino acid sequence identity between this enzyme, designated CTX-M-10, and the chromosomal β-lactamase ofKluyvera ascorbata was 81%.

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Distinct Regioselectivity of Fungal P450 Enzymes for Steroidal Hydroxylation

Applied and Environmental Microbiology ◽

10.1128/aem.01182-19 ◽

2019 ◽

Vol 85 (18) ◽

Cited By ~ 1

Author(s):

Wei Lu ◽

Jinhui Feng ◽

Xi Chen ◽

Yun-Juan Bao ◽

Yu Wang ◽

...

Keyword(s):

Amino Acid ◽

Pichia Pastoris ◽

Organic Synthesis ◽

Transcriptome Sequencing ◽

Amino Acid Sequence Identity ◽

Steroid Nucleus ◽

Content Type ◽

Whole Cells ◽

Sequence Identity ◽

High Amino Acid

ABSTRACT In this study, we identified two P450 enzymes (CYP5150AP3 and CYP5150AN1) from Thanatephorus cucumeris NBRC 6298 by combination of transcriptome sequencing and heterologous expression in Pichia pastoris. The biotransformation of 11-deoxycortisol and testosterone by Pichia pastoris whole cells coexpressing the cyp5150ap3 and por genes demonstrated that the CYP5150AP3 enzyme possessed steroidal 7β-hydroxylase activities toward these substrates, and the regioselectivity was dependent on the structures of steroidal compounds. CYP5150AN1 catalyzed the 2β-hydroxylation of 11-deoxycortisol. It is interesting that they display different regioselectivity of hydroxylation from that of their isoenzyme, CYP5150AP2, which possesses 19- and 11β-hydroxylase activities. IMPORTANCE The steroidal hydroxylases CYP5150AP3 and CYP5150AN1 together with the previously characterized CYP5150AP2 belong to the CYP5150A family of P450 enzymes with high amino acid sequence identity, but they showed completely different regioselectivities toward 11-deoxycortisol, suggesting the regioselectivity diversity of steroidal hydroxylases of CYP5150 family. They are also distinct from the known bacterial and fungal steroidal hydroxylases in substrate specificity and regioselectivity. Biocatalytic hydroxylation is one of the important transformations for the functionalization of steroid nucleus rings but remains a very challenging task in organic synthesis. These hydroxylases are useful additions to the toolbox of hydroxylase enzymes for the functionalization of steroids at various positions.

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Molecular Characterization of Genes Coding for Wild-Type and Sulfonylurea-Resistant Acetolactate Synthase in the Cyanobacterium Synechococcus PC C7942

Zeitschrift für Naturforschung C ◽

10.1515/znc-1990-0541 ◽

1990 ◽

Vol 45 (5) ◽

pp. 538-543 ◽

Cited By ~ 6

Author(s):

D. Friedberg ◽

J. Seijffers

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Amino Acid Sequence ◽

Molecular Characterization ◽

Amino Acid Level ◽

Acetolactate Synthase ◽

Mutant Gene ◽

Wild Type ◽

E Coli

We present here the isolation and molecular characterization of acetolactate synthase (ALS) genes from the cyanobacterium Synechococcus PCC7942 which specify a sulfonylurea-sensitive enzyme and from the sulfonylurea-resistant mutant SM3/20, which specify resistance to sulfonylurea herbicides. The ALS gene was cloned and mapped by complementation of an Escherichia coli ilv auxotroph that requires branched-chain amino acids for growth and lacks ALS activity. The cyanobacterial gene is efficiently expressed in this heterologous host. The ALS gene codes for 612 amino acids and shows high sequence homology (46%) at the amino acid level with ALS III of E. coli and with the tobacco ALS. The resistant phenotype is a consequence of proline to serine substitution in residue 115 of the deduced amino acid sequence. Functional expression of the mutant gene in wild-type Synechococcus and in E. coli confirmed that this amino-acid substitution is responsible for the resistance. Yet the deduced amino-acid sequence as compared with othjer ALS proteins supports the notion that the amino-acid context of the substitution is important for the resistance.

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