scholarly journals Characterization of Class V DyP-Type Peroxidase SaDyP1 from Streptomyces avermitilis and Evaluation of SaDyPs Expression in Mycelium

2021 ◽  
Vol 22 (16) ◽  
pp. 8683
Author(s):  
Kanako Sugawara ◽  
Toru Yoshida ◽  
Rena Hirashima ◽  
Ryoko Toriumi ◽  
Hotaka Akiyama ◽  
...  
Keyword(s):  
Amino Acid Sequence Identity ◽  
Streptomyces Avermitilis ◽  
Wild Type ◽  
Class V ◽  
Anthraquinone Dyes ◽  
Sequence Identity ◽  
High Amino Acid ◽  
Heme Peroxidases ◽  
Optimal Ph

DyP-type peroxidases are a family of heme peroxidases named for their ability to degrade persistent anthraquinone dyes. DyP-type peroxidases are subclassified into three classes: classes P, I and V. Based on its genome sequence, Streptomyces avermitilis, eubacteria, has two genes presumed to encode class V DyP-type peroxidases and two class I genes. We have previously shown that ectopically expressed SaDyP2, a member of class V, indeed has the characteristics of a DyP-type peroxidase. In this study, we analyzed SaDyP1, a member of the same class V as SaDyP2. SaDyP1 showed high amino acid sequence identity to SaDyP2, retaining a conserved GXXDG motif and catalytic aspartate. SaDyP1 degraded anthraquinone dyes, which are specific substrates of DyP-type peroxidases but not azo dyes. In addition to such substrate specificity, SaDyP1 showed other features of DyP-type peroxidases, such as low optimal pH. Furthermore, immunoblotting using an anti-SaDyP2 polyclonal antibody revealed that SaDyP1 and/or SaDyP2 is expressed in mycelia of wild-type S. avermitilis.

Biochemical characterization of a glucoamylase from Saccharomycopsis fibuligera R64

Biologia ◽  
2011 ◽  
Vol 66 (1) ◽  
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.

scholarly journals Petunia vein banding virus: Characterization of a New Tymovirus from Petunia × hybrida

Plant Disease ◽  
2000 ◽  
Vol 84 (7) ◽  
pp. 739-742 ◽  
Author(s):  
M. A. V. Alexandre ◽  
L. M. L. Duarte ◽  
E. B. Rivas ◽  
C. M. Chagas ◽  
M. M. Barradas ◽  
...  
Keyword(s):  
Nicotiana Benthamiana ◽  
Petunia Hybrida ◽  
Amino Acid Sequence Identity ◽  
Rio Grande Do Sul ◽  
Cytopathic Effects ◽  
Sequence Identity ◽  
Andean Potato ◽  
Plant Families ◽  
Nicandra Physalodes

Petunia plants from a nursery in the State of Rio Grande do Sul, Brazil, showed pronounced vein banding and contained isometric particles with diameters of approximately 45 and 30 nm. The larger ones apparently represent a caulimovirus, while the smaller ones, which included both empty shells and full particles, were identified as those of a new tymovirus for which we propose the name Petunia vein banding virus (PetVBV). Originally, PetVBV was transmitted only with difficulty to healthy petunia plants. However, from an experimentally infected petu-nia, it was later readily transmitted also to Nicotiana benthamiana and Nicandra physalodes, but not to other species in the Solanaceae or other plant families. It produces cytopathic effects typical for tymovirus infections. Its coat protein shows approximately 65% amino acid sequence identity with those of Eggplant mosaic and Andean potato latent viruses, to which it is also serologically more closely related than to any other tymoviruses.

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

2001 ◽  
Vol 45 (2) ◽  
pp. 616-620 ◽  
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%.

scholarly journals Distinct Regioselectivity of Fungal P450 Enzymes for Steroidal Hydroxylation

2019 ◽  
Vol 85 (18) ◽  
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.

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

2018 ◽  
Vol 62 (10) ◽  
Author(s):  
L. Dabos ◽  
A. B. Jousset ◽  
R. A. Bonnin ◽  
N. Fortineau ◽  
A. Zavala ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Amino Acid Sequence Identity ◽  
Biochemical Characterization ◽  
Content Type ◽  
Sequence Identity

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.

scholarly journals Two Novel Esterases from Deep-Sea Sediment Reveal Key Residues for Thermostability

2021 ◽  
Author(s):  
Yi Ding ◽  
Xiao-chen Yang ◽  
Ying-yi Huo ◽  
Shu-ling Jian ◽  
Xue-we Xu ◽  
...  
Keyword(s):  
Deep Sea ◽  
Amino Acid Sequence Identity ◽  
Metagenomic Library ◽  
Characteristic Analysis ◽  
Deep Sea Sediment ◽  
Sequence Identity ◽  
High Amino Acid ◽  
Application Potential ◽  
Key Residues ◽  
Family Iv

Abstract Thermostability is one of the major concerns in the industrial application of enzymes. In this study, two novel family IV esterases, Est2 and Est4, were identified from a deep-sea sediment metagenomic library. The two enzymes had high amino acid sequence identity (96%) with only twelve different residues. Characteristic analysis indicated that both enzymes shared most of the enzymatic properties, including optimum p-nitrophenyl substrates (p-nitrophenyl butyrate and hexanoate), temperature (40°C) and pH (7.0–8.0). Interestingly, Est2 showed higher thermostability at 50°C than Est4. Mutagenesis analysis of Est2 identified two out of twelve differential amino acids, Asp18 and Lys289, that were crucial for the thermostability of the enzymes. Asp18 determined both the thermostability and catalytic activity of Est2. Structural analysis showed that Asp18 was located at the cap domain of Est2 and might be involved in the mobility of the cap domain. Lys289, located at the surface of Est2, determined the discrepancy in the surface potential between the two enzymes. Our results provide inspiration for research on the thermostability of esterases and improve the application potential of deep sea-derived esterases in industrial production.

scholarly journals Role of a Ferredoxin Gene Cotranscribed with thenifHDK Operon in N2 Fixation and Nitrogenase “Switch-Off” of Azoarcus sp. Strain BH72

2001 ◽  
Vol 183 (12) ◽  
pp. 3752-3760 ◽  
Author(s):  
Tanja Egener ◽  
Dietmar E. Martin ◽  
Abhijit Sarkar ◽  
Barbara Reinhold-Hurek
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Deletion Mutant ◽  
Amino Acid Sequence Identity ◽  
Nitrogenase Activity ◽  
Gene Organization ◽  
Wild Type ◽  
Intracytoplasmic Membrane ◽  
Reversible Inactivation ◽  
Sequence Identity

ABSTRACT The endophytic diazotroph Azoarcus sp. strain BH72 is capable of infecting rice roots and of expressing the nitrogenase (nif) genes there. In order to study the genetic background for nitrogen fixation in strain BH72, the structural genes of nitrogenase (nifHDK) were cloned and sequenced. The sequence analysis revealed an unusual gene organization: downstream ofnifHDK, a ferredoxin gene (fdxN; 59% amino acid sequence identity to R. capsulatus FdxN) and open reading frames showing 52 and 36% amino acid sequence identity tonifY of Pseudomonas stutzeri A15 and ORF1 of Azotobacter vinelandii were located. Northern blot analysis, reverse transcriptase PCR and primer extension analysis revealed that these six genes are located on one transcript transcribed from a ς54-type promoter. Shorter transcripts sequentially missing genes of the 3′ part of the full-length mRNA were more abundantly detected. Mutational analyses suggested that FdxN is an important but not the essential electron donor for dinitrogenase reductase. An in-frame deletion of fdxN resulted in reduced growth rates (59% ± 9%) and nitrogenase activities (81%) in nitrogen-fixing pure cultures in comparison to the wild type. Nitrogenase activity was fully complemented in an fdxNmutant which carried a nifH promoter-drivenfdxN gene in trans. Also, in coculture with the ascomycete Acremonium alternatum, where strain BH72 develops intracytoplasmic membrane stacks, the nitrogenase activity in the fdxN deletion mutant was decreased to 56% of the wild-type level. Surprisingly, the fdxNdeletion also had an effect on the rapid “switch-off” of nitrogenase activity in response to ammonium. Wild-type strain BH72 and the deletion mutant complemented with fdxN intrans showed a rapid reversible inactivation of acetylene reduction, while the deletion mutant did not cease to reduce acetylene. In concordance with the hypothesis that changes in the redox state of NifH or electron flux towards nitrogenase may be involved in the mechanism of physiological nitrogenase switch-off, our results suggest that the ferredoxin may be a component involved in this process.

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

1999 ◽  
Vol 10 (1) ◽  
pp. 119-134 ◽  
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.

scholarly journals Tropomyosin is essential in yeast, yet the TPM1 and TPM2 products perform distinct functions.

1995 ◽  
Vol 128 (3) ◽  
pp. 383-392 ◽  
Author(s):  
B Drees ◽  
C Brown ◽  
B G Barrell ◽  
A Bretscher
Keyword(s):  
Sequence Analysis ◽  
Wild Type ◽  
Purification And Characterization ◽  
Reading Frame ◽  
Over Expression ◽  
Sequence Identity ◽  
Bona Fide ◽  
Essential Function

Sequence analysis of chromosome IX of Saccharomyces cerevisiae revealed an open reading frame of 166 residues, designated TPM2, having 64.5% sequence identity to TPM1, that encodes the major form of tropomyosin in yeast. Purification and characterization of Tpm2p revealed a protein with the characteristics of a bona fide tropomyosin; it is present in vivo at about one sixth the abundance of Tpm1p. Biochemical and sequence analysis indicates that Tpm2p spans four actin monomers along a filament, whereas Tpmlp spans five. Despite its shorter length, Tpm2p can compete with Tpm1p for binding to F-actin. Over-expression of Tpm2p in vivo alters the axial budding of haploids to a bipolar pattern, and this can be partially suppressed by co-over-expression of Tpm1p. This suggests distinct functions for the two tropomyosins, and indicates that the ratio between them is important for correct morphogenesis. Loss of Tpm2p has no detectable phenotype in otherwise wild type cells, but is lethal in combination with tpm1 delta. Over-expression of Tpm2p does not suppress the growth or cell surface targeting defects associated with tpm1 delta, so the two tropomyosins must perform an essential function, yet are not functionally interchangeable. S. cerevisiae therefore provides a simple system for the study of two tropomyosins having distinct yet overlapping functions.

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