scholarly journals The laterally acquired GH5 ZgEngAGH5_4 from the marine bacterium Zobellia galactanivorans is dedicated to hemicellulose hydrolysis

2018 ◽  
Vol 475 (22) ◽  
pp. 3609-3628 ◽  
Author(s):  
Jonathan Dorival ◽  
Sophie Ruppert ◽  
Melissa Gunnoo ◽  
Adam Orłowski ◽  
Maylis Chapelais-Baron ◽  
...  
Keyword(s):  
Heterotrophic Bacteria ◽  
Carbon Sources ◽  
Binding Pocket ◽  
Catalytic Triad ◽  
Marine Macroalgae ◽  
Amino Acid Residues ◽  
Glucose Binding ◽  
Marine Heterotrophic Bacteria ◽  
Binding Subsites

Cell walls of marine macroalgae are composed of diverse polysaccharides that provide abundant carbon sources for marine heterotrophic bacteria. Among them, Zobellia galactanivorans is considered as a model for studying algae–bacteria interactions. The degradation of typical algal polysaccharides, such as agars or alginate, has been intensively studied in this model bacterium, but the catabolism of plant-like polysaccharides is essentially uncharacterized. Here, we identify a polysaccharide utilization locus in the genome of Z. galactanivorans, induced by laminarin (β-1,3-glucans), and containing a putative GH5 subfamily 4 (GH5_4) enzyme, currently annotated as a endoglucanase (ZgEngAGH5_4). A phylogenetic analysis indicates that ZgEngAGH5_4 was laterally acquired from an ancestral Actinobacteria. We performed the biochemical and structural characterization of ZgEngAGH5_4 and demonstrated that this GH5 is, in fact, an endo-β-glucanase, most active on mixed-linked glucan (MLG). Although ZgEngAGH5_4 and GH16 lichenases both hydrolyze MLG, these two types of enzymes release different series of oligosaccharides. Structural analyses of ZgEngAGH5_4 reveal that all the amino acid residues involved in the catalytic triad and in the negative glucose-binding subsites are conserved, when compared with the closest relative, the cellulase EngD from Clostridium cellulovorans, and some other GH5s. In contrast, the positive glucose-binding subsites of ZgEngAGH5_4 are different and this could explain the preference for MLG, with respect to cellulose or laminarin. Molecular dynamics computer simulations using different hexaoses reveal that the specificity for MLG occurs through the +1 and +2 subsites of the binding pocket that display the most important differences when compared with the structures of other GH5_4 enzymes.

scholarly journals Determination of three amino acids causing alteration of proteolytic activities of staphylococcal glutamyl endopeptidases

2009 ◽  
Vol 390 (3) ◽  
Author(s):  
Takayuki K. Nemoto ◽  
Toshio Ono ◽  
Yu Shimoyama ◽  
Shigenobu Kimura ◽  
Yuko Ohara-Nemoto
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Protease Activity ◽  
Binding Pocket ◽  
Catalytic Triad ◽  
Amino Acid Residues ◽  
Proteolytic Activities ◽  
Hydrophobic Amino Acids ◽  
The Difference

Abstract Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus warneri secrete glutamyl endopeptidases, designated GluV8, GluSE, and GluSW, respectively. The order of their protease activities is GluSE<GluSW<<GluV8. In the present study, we investigated the mechanism that causes these differences. Expression of chimeric proteins between GluV8 and GluSE revealed that the difference is primarily attributed to amino acid residues 170–195, which define the intrinsic protease activity, and additionally to residues 119–169, which affect the proteolytic sensitivity. Among nine substitutions present in residues 170–195 of the three proteases, the substitutions at positions 185, 188, and 189 were responsible for the changes in their activities, and the combination of W185, V188, and P189, which naturally occurs in GluV8, exerts the highest protease activity. W185 and P189 were indispensable for full activity, but V188 could be replaced by hydrophobic amino acids. These three amino acid residues appear to create a substrate-binding pocket together with the catalytic triad and the N-terminal V1, and therefore define the K m values of the proteases. We also describe a method to produce a chimeric form of GluSE and GluV8 that is resistant to proteolysis, and therefore possesses 4-fold higher activity than the wild-type recombinant GluV8.

Characterization of an opsin gene from the ascomycete Leptosphaeria maculans

Genome ◽  
2001 ◽  
Vol 44 (2) ◽  
pp. 167-171 ◽  
Author(s):  
Alexander Idnurm ◽  
Barbara J Howlett
Keyword(s):  
Leptosphaeria Maculans ◽  
Brassica Species ◽  
Binding Pocket ◽  
Opsin Gene ◽  
Amino Acid Residues ◽  
Blackleg Disease ◽  
Type Locus ◽  
Cloned Gene ◽  
Retinal Binding Pocket

An opsin gene (ops) has been characterized from Leptosphaeria maculans, the ascomycete that causes blackleg disease of Brassica species. This is the second opsin identified outside the archaeal and animal kingdoms. The gene encodes a predicted protein with high similarity (70.3%) and identity (53.3%) to the nop-1 opsin of another ascomycete Neurospora crassa. The L. maculans opsin also has identical amino acid residues in 20 of the 22 residues in the retinal-binding pocket of archaeal opsins. Opsin, on the fourth largest chromosome of L. maculans and 22 cM from the mating type locus, is the first cloned gene to be mapped in L. maculans. Opsin is transcribed at high levels in mycelia grown in the presence and absence of light; this pattern is in contrast with that of the N. crassa opsin, which is transcribed only in the light.Key words: opsin, Phoma lingam, Brassica napus.

scholarly journals Cloning and Characterization of Lipase Gene from a Local Isolate of Pseudoxanthomonas sp.

2017 ◽  
Vol 14 (2) ◽  
pp. 503-507 ◽  
Author(s):  
Yogi Yopa Kristia ◽  
Syifa F Syihab ◽  
Akhmaloka Akhmaloka
Keyword(s):  
Crystal Structure ◽  
3D Structure ◽  
Catalytic Triad ◽  
Catalytic Residue ◽  
Pseudomonas Sp ◽  
Amino Acid Residues ◽  
Chromosomal Dna ◽  
Lipase Gene

ABSTRACT: Lipase gene from Pseudoxanthomonas sp. was cloned through in vitro amplification from total chromosomal DNA. The gene was sequenced and characterized, coding for 312 amino acid residues. Homological analysis showed that the gene has 98% similarity to lipolytic gene from Uncultured Pseudomonas sp (GenBank No. AKA58891.1). Further analysis appeared that the sequences showed similar unique motifs of lipase sub-family I.1, such as pentapeptide (GHSHG) motif, tetrapeptide (GMLG) motif, and catalytic triad. In additional, 3D structure analysis based on crystal structure of Pseudomonas aeruginose (PDB ID 1ex9) showed that both structure of lipases are similar except on the conformation of catalytic residue of His277 showing to shift more far away compared to that the control.

Identification and characterization of ace1-type acetylcholinesterase in insecticide-resistant and -susceptible Propylaea japonica (Thunberg)

2017 ◽  
Vol 108 (2) ◽  
pp. 253-262 ◽  
Author(s):  
M.M. Wang ◽  
L.Y. Xing ◽  
Z.W. Ni ◽  
G. Wu
Keyword(s):  
Amino Acids ◽  
Disulfide Bonds ◽  
Aromatic Amino Acids ◽  
Binding Pocket ◽  
Catalytic Triad ◽  
Determining Factors ◽  
Omega Loop ◽  
Predacious Species ◽  
First Time

AbstractCharacterization and gene cloning of acetylecholinesterase (AChE) in the insecticide-resistant (R) and -susceptible (S) insects have been reported in the past. However, the studies focused mostly on herbivorous pests, rather than predacious species, such as ladybird beetles. Using R and S Propylaea japonica (thunberg), a full-length cDNA sequence (2928 bp) of the ace1-type AChE gene was determined for the first time. The ace1 encoding a protein of 645 amino acids contained typical conserved motifs, such as FGESAG domains, catalytic triad, acyl pocket, oxyanino hole, choline binding site, peripheral anionic site, omega loop and conserved aromatic residues. R P. japonica displayed 50-times greater resistance to chlorpyrifos or mathamidophos with a significantly lower AChE sensitivity to paraoxon, malaoxon, chlorpyrifos or methamidophos than its S counterpart. Five amino acids in the ace1 of R P. japonica differed from those found in S P. japonica. One of them, F358S, located in the acyl-binding pocket, might play a crucial role in the resistance of the insect to organophosphates (OPs). Whereas, K493E and I538V, which were close to some of the conserved aromatic amino acids (i.e., H509, Y511, and W499) in the gorge, and G571R and T576A near C593 that formed the disulfide bonds with C471, might also involve in the change of insecticide resistance in P. japonica. AChE insensitivity and amino acid replacements, particularly F358S, might be the determining factors in the alteration of OPs-resistance in P. japonica.

scholarly journals Characterization of a glycolipid glycosyltransferase with broad substrate specificity from the marine bacterium Candidatus Pelagibacter sp. HTCC7211

2021 ◽  
Author(s):  
Tao Wei ◽  
Caimeng Zhao ◽  
Mussa Quareshy ◽  
Nan Wu ◽  
Shen Huang ◽  
...  
Keyword(s):  
Marine Bacterium ◽  
Heterotrophic Bacteria ◽  
Homology Modelling ◽  
Catalytic Efficiency ◽  
Bioactive Molecules ◽  
Marine Microbes ◽  
Synthesis Mechanism ◽  
Marine Heterotrophic Bacteria ◽  
Sar11 Clade

In the marine environment, phosphorus availability significantly affects the lipid composition in many cosmopolitan marine heterotrophic bacteria, including members of the SAR11 clade and the Roseobacter clade. Under phosphorus stress conditions, non-phosphorus sugar-containing glycoglycerolipids are substitutes for phospholipids in these bacteria. Although these glycoglycerolipids play an important role as surrogates for phospholipids under phosphate deprivation, glycoglycerolipid synthases in marine microbes are poorly studied. In the present study, we biochemically characterized a glycolipid glycosyltransferase (GTcp) from the marine bacterium Candidatus Pelagibacter sp. HTCC7211, a member of the SAR11 clade. Our results showed that GTcp is able to act as a multifunctional enzyme by synthesizing different glycoglycerolipids with UDP-glucose, UDP-galactose, or UDP-glucuronic acid as sugar donors and diacylglycerol as the acceptor. Analyses of enzyme kinetic parameters demonstrated that Mg2+ notably changes the enzyme's affinity for UDP-glucose, which improves its catalytic efficiency. Homology modelling and mutational analyses revealed binding sites for the sugar donor and the diacylglycerol lipid acceptor, which provided insights into the retaining mechanism of GTcp with its GT-B fold. A phylogenetic analysis showed that GTcp and its homologs form a group in the GT4 glycosyltransferase family. These results not only provide new insights into the glycoglycerolipid synthesis mechanism in lipid remodelling, but also describe an efficient enzymatic tool for future synthesis of bioactive molecules.

scholarly journals Isolation and characterization of the chicken trypsinogen gene family

1995 ◽  
Vol 307 (2) ◽  
pp. 471-479 ◽  
Author(s):  
K Wang ◽  
L Gan ◽  
I Lee ◽  
L Hood
Keyword(s):  
Amino Acid ◽  
Gene Family ◽  
Amino Acid Level ◽  
Structural Features ◽  
Catalytic Triad ◽  
Amino Acid Residues ◽  
Isolation And Characterization ◽  
Anionic Trypsin ◽  
Cationic Trypsin

Based on genomic Southern hybridizations and cDNA sequence analyses, the chicken trypsinogen gene family can be divided into two multi-member subfamilies, a six-member trypsinogen I subfamily which encodes the cationic trypsin isoenzymes and a three-member trypsinogen II subfamily which encodes the anionic trypsin isoenzymes. The chicken cDNA and genomic clones containing these two subfamilies were isolated and characterized by DNA sequence analysis. The results indicated that the chicken trypsinogen genes encoded a signal peptide of 15 to 16 amino acid residues, an activation peptide of 9 to 10 residues and a trypsin of 223 amino acid residues. The chicken trypsinogens contain all the common catalytic and structural features for trypsins, including the catalytic triad His, Asp and Ser and the six disulphide bonds. The trypsinogen I and II subfamilies share approximately 70% sequence identity at the nucleotide and amino acid level. The sequence comparison among chicken trypsinogen subfamily members and trypsin sequences from other species suggested that the chicken trypsinogen genes may have evolved in coincidental or concerted fashion.

scholarly journals The levansucrase and inulosucrase enzymes of Lactobacillus reuteri 121 catalyse processive and non-processive transglycosylation reactions

Microbiology ◽  
2006 ◽  
Vol 152 (4) ◽  
pp. 1187-1196 ◽  
Author(s):  
Lukasz K. Ozimek ◽  
Slavko Kralj ◽  
Marc J. E. C. van der Maarel ◽  
Lubbert Dijkhuizen
Keyword(s):  
Amino Acid ◽  
Catalytic Mechanism ◽  
Lactobacillus Reuteri ◽  
Amino Acid Residues ◽  
Reaction Products ◽  
Detailed Characterization ◽  
Amino Acid Sequence Level ◽  
Chain Polymerization ◽  
Binding Subsites

Bacterial fructosyltransferase (FTF) enzymes synthesize fructan polymers from sucrose. FTFs catalyse two different reactions, depending on the nature of the acceptor, resulting in: (i) transglycosylation, when the growing fructan chain (polymerization), or mono- and oligosaccharides (oligosaccharide synthesis), are used as the acceptor substrate; (ii) hydrolysis, when water is used as the acceptor. Lactobacillus reuteri 121 levansucrase (Lev) and inulosucrase (Inu) enzymes are closely related at the amino acid sequence level (86 % similarity). Also, the eight amino acid residues known to be involved in catalysis and/or sucrose binding are completely conserved. Nevertheless, these enzymes differ markedly in their reaction and product specificities, i.e. in β(2→6)- versus β(2→1)-glycosidic-bond specificity (resulting in levan and inulin synthesis, respectively), and in the ratio of hydrolysis versus transglycosylation activities [resulting in glucose and fructooligosaccharides (FOSs)/polymer synthesis, respectively]. The authors report a detailed characterization of the transglycosylation reaction products synthesized by the Lb. reuteri 121 Lev and Inu enzymes from sucrose and related oligosaccharide substrates. Lev mainly converted sucrose into a large levan polymer (processive reaction), whereas Inu synthesized mainly a broad range of FOSs of the inulin type (non-processive reaction). Interestingly, the two FTF enzymes were also able to utilize various inulin-type FOSs (1-kestose, 1,1-nystose and 1,1,1-kestopentaose) as substrates, catalysing a disproportionation reaction; to the best of our knowledge, this has not been reported for bacterial FTF enzymes. Based on these data, a model is proposed for the organization of the sugar-binding subsites in the two Lb. reuteri 121 FTF enzymes. This model also explains the catalytic mechanism of the enzymes, and differences in their product specificities.

scholarly journals Identification and characterization of amphibian SLC26A5 using RNA-Seq

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Zhongying Wang ◽  
Qixuan Wang ◽  
Hao Wu ◽  
Zhiwu Huang
Keyword(s):  
Amino Acid ◽  
Inner Ear ◽  
Hair Cells ◽  
Rana Catesbeiana ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Rna Seq ◽  
American Bullfrog ◽  
Frog Species

Abstract Background Prestin (SLC26A5) is responsible for acute sensitivity and frequency selectivity in the vertebrate auditory system. Limited knowledge of prestin is from experiments using site-directed mutagenesis or domain-swapping techniques after the amino acid residues were identified by comparing the sequence of prestin to those of its paralogs and orthologs. Frog prestin is the only representative in amphibian lineage and the studies of it were quite rare with only one species identified. Results Here we report a new coding sequence of SLC26A5 for a frog species, Rana catesbeiana (the American bullfrog). In our study, the SLC26A5 gene of Rana has been mapped, sequenced and cloned successively using RNA-Seq. We measured the nonlinear capacitance (NLC) of prestin both in the hair cells of Rana’s inner ear and HEK293T cells transfected with this new coding gene. HEK293T cells expressing Rana prestin showed electrophysiological features similar to that of hair cells from its inner ear. Comparative studies of zebrafish, chick, Rana and an ancient frog species showed that chick and zebrafish prestin lacked NLC. Ancient frog’s prestin was functionally different from Rana. Conclusions We mapped and sequenced the SLC26A5 of the Rana catesbeiana from its inner ear cDNA using RNA-Seq. The Rana SLC26A5 cDNA was 2292 bp long, encoding a polypeptide of 763 amino acid residues, with 40% identity to mammals. This new coding gene could encode a functionally active protein conferring NLC to both frog HCs and the mammalian cell line. While comparing to its orthologs, the amphibian prestin has been evolutionarily changing its function and becomes more advanced than avian and teleost prestin.

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