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

2018 ◽  
Vol 85 (3) ◽  
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.

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

2014 ◽  
Vol 81 (1) ◽  
pp. 272-281 ◽  
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.

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

2011 ◽  
Vol 56 (2) ◽  
pp. 666-671 ◽  
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.

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

2018 ◽  
Author(s):  
Laura L. Lee ◽  
William S. Hart ◽  
Vladimir V. Lunin ◽  
Markus Alahuhta ◽  
Yannick J. Bomble ◽  
...  
Keyword(s):  
Microcrystalline Cellulose ◽  
Binding Proteins ◽  
Hot Springs ◽  
Elevated Temperatures ◽  
Plant Biomass ◽  
Three Dimensional ◽  
Carbohydrate Binding ◽  
Additional Information ◽  
Carbohydrate Binding Modules ◽  
Cellulose Binding

AbstractGenomes of extremely thermophilic Caldicellulosiruptor species encode novel cellulose binding proteins, tāpirins, located proximate to the type IV pilus locus. Previously, the C-terminal domain of a tāpirin (Calkro_0844) from Caldicellulosiruptor kronotskyensis was shown to be structurally unique and have a cellulose binding affinity akin to family 3 carbohydrate binding modules (CBM3). 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, 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 to associate closely with biomass to capture glucans released from lignocellulose by cellulases produced in Caldicellulosiruptor communities. Three-dimensional structures of the C-terminal binding regions of Calhy_0908 and Calkr_0826 were closely related to Calkro_0844, despite the fact that their amino acid sequence identities compared to Calkro_0844 were only 16% and 36%, respectively. Unlike the parent strain, C. bescii mutants lacking the tāpirin genes did not bind to cellulose following short-term incubation, reinforcing the significance of these proteins in cell association with plant biomass. Given the scarcity of carbohydrates in neutral terrestrial hot springs, tāpirins likely help cells scavenge carbohydrates from lignocellulose to support growth and survival of Caldicellulosiruptor species.ImportanceMechanisms 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 Caldicellulosiruptor species interact with microcrystalline cellulose and here additional information about the diversity of these proteins across the genus is provided, including three dimensional structural comparisons.

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 cDNA nucleotide sequence encoding the ZPC protein of Australian hydromyine rodents: a novel sequence of the putative sperm-combining site within the family Muridae

Zygote ◽  
2002 ◽  
Vol 10 (4) ◽  
pp. 291-299 ◽  
Author(s):  
Christine A. Swann ◽  
Rory M. Hope ◽  
William G. Breed
Keyword(s):  
Amino Acid ◽  
Nucleotide Sequence ◽  
Amino Acid Sequence ◽  
Cdna Sequence ◽  
Amino Acid Sequence Identity ◽  
Sequence Data ◽  
Coding Region ◽  
Cdna Sequences ◽  
Sequence Identity ◽  
Murid Rodents

This comparative study of the cDNA sequence of the zona pellucida C (ZPC) glycoprotein in murid rodents focuses on the nucleotide and amino acid sequence of the putative sperm-combining site. We ask the question: Has divergence evolved in the nucleotide sequence of ZPC in the murid rodents of Australia? Using RT-PCR and (RACE) PCR, the complete cDNA coding region of ZPC in the Australian hydromyine rodents Notomys alexis and Pseudomys australis, and a partial cDNA sequence from a third hydromyine rodent, Hydromys chrysogaster, has been determined. Comparison between the cDNA sequences of the hydromyine rodents reveals that the level of amino acid sequence identity between N. alexis and P. australis is 96%, whereas that between the two species of hydromyine rodents and M. musculus and R. norvegicus is 88% and 87% respectively. Despite being reproductively isolated from each other, the three species of hydromyine rodents have a 100% level of amino acid sequence identity at the putative sperm-combining site. This finding does not support the view that this site is under positive selective pressure. The sequence data obtained in this study may have important conservation implications for the dissemination of immunocontraception directed against M. musculus using ZPC antibodies.

scholarly journals Ribosomal proteins L34 and S29 of amphioxus Branchiostoma belcheri tsingtauense: cDNAs cloning and gene copy number.

2005 ◽  
Vol 52 (4) ◽  
pp. 857-862 ◽  
Author(s):  
Lina Liu ◽  
Shicui Zhang ◽  
Zhenhui Liu ◽  
Hongyan Li ◽  
Mei Liu ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Ribosomal Proteins ◽  
Amino Acid Sequence Identity ◽  
Fruit Fly ◽  
Calculated Molecular Mass ◽  
Sequence Identity ◽  
Acid Protein ◽  
Branchiostoma Belcheri ◽  
Amino Acid Protein

The complete cDNA and deduced amino-acid sequences of ribosomal proteins L34 (AmphiL34) and S29 (AmphiS29) from the amphioxus Branchiostoma belcheri tsingtauense were identified in this study. The AmphiL34 cDNA is 435 nucleotides in length and encodes a 118 amino-acid protein with calculated molecular mass of 13.6 kDa. It shares 53.6-67.5% amino-acid sequence identity with its eukaryotic counterparts including human, mouse, rat, pig, frog, catfish, fruit fly, mosquito, armyworm, nematode and yeast. The AmphiS29 cDNA comprises 453 nucleotides and codes for a 56 amino-acid protein with a calculated molecular mass of 6.6 kDa. It shows 66.1-78.6% amino-acid sequence identity to eukaryotic S29 proteins from human, mouse, rat, pig, zebrafish, seahorse, fruit fly, nematode, sea hare and yeast. AmphiL34 contains a putative nucleolar localization signal, while AmphiS29 has a zinc finger-like domain. A phylogenetic tree deduced from the conserved sequences of AmphiL34 and AmphiS29 and other known counterparts indicates that the positions of AmphiL34/AmphiS29 are intermediate between the vertebrate and invertebrate L34/S29. Southern blot analysis demonstrates the presence of one copy of the L34 gene and 2-3 copies of the S29 gene in the genome of the amphioxus B. belcheri tsingtauense. This is in sharp contrast to the existence of 7-9 copies of the L34 gene and 14-17 copies of the S29 gene in the rat genome. These date suggest that housekeeping genes like AmphiL34 and AmphiS29 have undergone large-scale duplication in the chordate lineage.

scholarly journals First Report of Beet soilborne virus in Poland

Plant Disease ◽  
2006 ◽  
Vol 90 (1) ◽  
pp. 112-112 ◽  
Author(s):  
N. Borodynko ◽  
B. Hasiów ◽  
H. Pospieszny
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Sugar Beet ◽  
Amino Acid Sequence Identity ◽  
Chenopodium Quinoa ◽  
Enzyme Linked Immunosorbent Assay ◽  
First Report ◽  
Sequence Identity ◽  
Pcr Products ◽  
Soilborne Viruses

Beet necrotic yellow vein virus (BNYVV), the casual agent of rhizomania disease, was identified in sugar beet plants from several fields in the Wielkopolska Region of Poland (1). In greenhouse studies, sugar beets were grown in the soil from one of these fields to bait soilborne viruses. Of 200 sugar beet plants, three developed symptoms of vein clearing, vein banding, and mosaic. Crude sap from symptomatic plants was used for mechanical inoculation of various plants species. In Chenopodium quinoa, C. amaranticolor, and Tetragonia expansa only local lesions were observed. Electron microscope examination of negatively stained leaf-dip preparations from symptomatic sugar beet plants showed a mixture of rod-shape particles from 70 to 400 nm long. Using double-antibody sandwich enzyme-linked immunosorbent assay tests, two symptomatic sugar beet plants gave positive reactions with antiserum against BNYVV (Bio-Rad, Hercules, CA) and a third plant gave a positive reaction with antisera against BNYVV and Beet soilborne virus (BSBV). Total RNA was extracted from roots and leaves of the symptomatic plants and used in a multiplex reverse transcription-polymerase chain reaction (mRT-PCR) assay. Specific primers were designed to amplify a fragment of the RNA1 for BSBV and RNA2 for BNYVV and Beet virus Q (BVQ) (2). Two mRT-PCR products amplified with the primers specific to BNYVV and BSBV were obtained and sequenced. A 274-nt amplicon sequence (GenBank Accession No. DQ012156) had 98% nucleotide sequence identity with the German BNYVV isolate F75 (GenBank Accession No. AF19754) and a 376-nt amplicon sequence (GenBank Accession No. AY999690) had 98% nucleotide and 98% amino acid sequence identity with the German BSBV isolate (GenBank Accession No. Z97873). The Polish BSBV isolate had 88% nucleotide and 62% amino acid sequence identity with BVQ, another pomovirus (GenBank Accession No. AJ 223596 formerly known as serotype Wierthe of BSBV (2). In 2005, mRT-PCR was used on samples collected from two fields of the Wielkopolska Region. Of 15 tested sugar beet plants, 12 gave positive reactions with primers specific for BSBV and nine with primers specific to BNYVV. To our knowledge, this is first report of BSBV in Poland. In Europe, BSBV was previously reported in England, the Netherlands, Belgium, Sweden, Germany, France, and Finland (2,3). References: (1) M. Jezewska and J. Piszczek. Phytopathol. Polonica, 21:165, 2001. (2) A. Maunier et al. Appl. Environ. Microbiol. 69:2356, 2003. (3) C. M. Rush and G. B. Heidel. Plant Dis. 79:868, 1995.

Characterization of a novel heterodimeric cathepsin L-like protease and cDNA encoding the catalytic subunit of the protease in embryos of Artemia franciscana

2001 ◽  
Vol 79 (1) ◽  
pp. 43-56 ◽  
Author(s):  
Ann M Butler ◽  
Andrea L Aiton ◽  
Alden H Warner
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Amino Acid Sequence Identity ◽  
Single Gene ◽  
Cathepsin L ◽  
Cysteine Proteases ◽  
Artemia Franciscana ◽  
Catalytic Subunit ◽  
Mature Protein ◽  
Sequence Identity

Embryos and larvae of the brine shrimp, Artemia franciscana, contain a novel cathepsin L-like cysteine protease (ACP) composed of 28.5- and 31.5-kDa subunits. Both subunits of the ACP are glycosylated, and seven isoforms of the protease were identified by isoelectric focusing with pI values ranging from 4.6 to 6.2. Several clones containing sequences coding for the 28.5-kDa subunit of the ACP were isolated from an Artemia embryo cDNA library in lambda ZAP II. One clone of 1229 bp, with an open reading frame of 1014 bp, was sequenced and found to contain 50-65% amino acid sequence identity with several members of the cathepsin L subfamily of cysteine proteases. The mature protein predicted from this sequence consisted of 217 amino acids with a mass of 23.5 kDa prior to post-translational modifications. The mature protein showed 68.6% amino acid sequence identity with human cathepsin L and 73.9% identity with cathepsin L-like proteases from Sarcophaga. peregrina and Drosophila melanogaster. The full-length cDNA clone analyzed in this study (pCP-3b) was renamed AFCATL1 (A. franciscana Cathepsin L1) and the sequence has been deposited in the Genbank database, accession number AF147207. Northern blot analyses identified a single transcript of about 1.4 kb in both embryos and young larvae of Artemia. Southern blot analyses of Artemia genomic DNA treated with various restriction endonucleases indicated a single gene for the ACP. The catalytic subunit of the ACP was tightly associated with a 31.5-kDa protein, which may localize the protease to nonlysosomal sites in embryos and larvae.Key words: cathepsin L, proteases, embryos, development, Artemia.

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