Biochemical characterization and mutational analysis of a novel flap endonuclease 1 from Thermococcus barophilus Ch5

2022 ◽  
pp. 106154
Author(s):  
Tan Lin ◽  
Likui Zhang ◽  
Donghao Jiang ◽  
Leilei Wu ◽  
Kaige Chen ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Mutational Analysis ◽  
Flap Endonuclease 1

Biochemical Characterization and Mutational Analysis of a Lactone Hydrolase from Phialophora americana

2019 ◽  
Vol 68 (8) ◽  
pp. 2570-2577
Author(s):  
Xinrui Yu ◽  
Tao Tu ◽  
Huiying Luo ◽  
Huoqing Huang ◽  
Xiaoyun Su ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Mutational Analysis

Biochemistry and site-directed mutational analysis of Δ7-sterol-C5(6)-desaturase

2000 ◽  
Vol 28 (6) ◽  
pp. 799-803 ◽  
Author(s):  
A. Rahier ◽  
P. Benveniste ◽  
T. Husselstein ◽  
M. Taton
Keyword(s):  
Biochemical Characterization ◽  
Mutational Analysis ◽  
Enzyme System ◽  
Conserved Residues ◽  
Catalytic Role ◽  
Membrane Bound ◽  
Haem Iron ◽  
Oxygen Site

This report describes recent work on the process of desaturation at C5(6) of sterol precursors in plants. Biochemical characterization of the plant Δ7-sterol C5(6)-desaturase (5-DES) indicates that the enzyme system involved shows important similarities to the soluble and membrane-bound non-haem iron desaturases found in eukaryotes, including cyanide and hydrophobic chelators sensitivity, CO resistance and a requirement for exogenous reductant and molecular oxygen. Site-directed mutational analysis of highly conserved residues in 5-DES indicated that eight histidine residues from three histidine-rich motifs were essential for the catalysis, possibly by providing the ligands for a putative Fe centre. This mutational analysis also revealed the catalytic role of the functionally conserved Thr-114.

Biochemical characterization and mutational analysis of alanine racemase from Clostridium perfringens

2019 ◽  
Vol 128 (2) ◽  
pp. 149-155
Author(s):  
Muhammad Israr ◽  
Guoping Lv ◽  
Shujing Xu ◽  
Yunhe Li ◽  
Shengting Ding ◽  
...  
Keyword(s):  
Clostridium Perfringens ◽  
Biochemical Characterization ◽  
Mutational Analysis ◽  
Alanine Racemase

Biochemical characterization and mutational analysis of the mononuclear non-haem Fe2+ site in Dke1, a cupin-type dioxygenase from Acinetobacter johnsonii

2009 ◽  
Vol 418 (2) ◽  
pp. 403-411 ◽  
Author(s):  
Stefan Leitgeb ◽  
Grit D. Straganz ◽  
Bernd Nidetzky
Keyword(s):  
Metal Binding ◽  
Biochemical Characterization ◽  
Mutational Analysis ◽  
Wild Type ◽  
Acinetobacter Johnsonii ◽  
Metal Binding Sites ◽  
Oxidative Inactivation ◽  
Bivalent Metal Ions ◽  
Catalytic Metal ◽  
Cupin Superfamily

β-Diketone-cleaving enzyme Dke1 is a homotetrameric Fe2+-dependent dioxygenase from Acinetobacter johnsonii. The Dke1 protomer adopts a single-domain β-barrel fold characteristic of the cupin superfamily of proteins and features a mononuclear non-haem Fe2+ centre where a triad of histidine residues, His-62, His-64 and His-104, co-ordinate the catalytic metal. To provide structure–function relationships for the peculiar metal site of Dke1 in relation to the more widespread 2-His-1-Glu/Asp-binding site for non-haem Fe2+, we replaced each histidine residue individually with glutamate and asparagine and compared binding of Fe2+ and four non-native catalytically inactive metals with purified apo-forms of wild-type and mutant enzymes. Results from anaerobic equilibrium microdialysis (Fe2+) and fluorescence titration (Fe2+, Cu2+, Ni2+, Mn2+ and Zn2+) experiments revealed the presence of two broadly specific metal-binding sites in native Dke1 that bind Fe2+ with a dissociation constant (Kd) of 5 μM (site I) and ∼0.3 mM (site II). Each mutation, except for the substitution of asparagine for His-104, disrupted binding of Fe2+, but not that of the other bivalent metal ions, at site I, while leaving metal binding at site II largely unaffected. Dke1 mutants harbouring glutamate substitutions were completely inactive and not functionally complemented by external Fe2+. The Fe2+ catalytic centre activity (kcat) of mutants with asparagine substitution of His-62 and His-104 was decreased 140- and 220-fold respectively, compared with the kcat value of 8.5 s−1 for the wild-type enzyme in the reaction with pentane-2,4-dione. The H64N mutant was not catalytically competent, except in the presence of external Fe2+ (1 mM) which elicited about 1/1000 of wild-type activity. Therefore co-ordination of Fe2+ by Dke1 requires an uncharged metallocentre, and three histidine ligands are needed for the assembly of a fully functional catalytic site. Oxidative inactivation of Dke1 was shown to involve conversion of enzyme-bound Fe2+ into Fe3+, which is then released from the metal centre.

scholarly journals Biochemical characterization of the cyclooxygenase enzyme in penaeid shrimp

PLoS ONE ◽  
2021 ◽  
Vol 16 (4) ◽  
pp. e0250276
Author(s):  
Punsa Tobwor ◽  
Pacharawan Deenarn ◽  
Thapanee Pruksatrakul ◽  
Surasak Jiemsup ◽  
Suganya Yongkiettrakul ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Biochemical Characterization ◽  
Mutational Analysis ◽  
Penaeus Monodon ◽  
Penaeid Shrimp ◽  
Prostaglandin E ◽  
Catalytic Function ◽  
Enzymatic Function ◽  
Glycosylation Sites ◽  
Prostaglandin F

Cyclooxygenase (COX) is a two-step enzyme that converts arachidonic acid into prostaglandin H2, a labile intermediate used in the production of prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α). In vertebrates and corals, COX must be N-glycosylated on at least two asparagine residues in the N-(X)-S/T motif to be catalytically active. Although COX glycosylation requirement is well-characterized in many species, whether crustacean COXs require N-glycosylation for their enzymatic function have not been investigated. In this study, a 1,842-base pair cox gene was obtained from ovarian cDNA of the black tiger shrimp Penaeus monodon. Sequence analysis revealed that essential catalytic residues and putative catalytic domains of P. monodon COX (PmCOX) were well-conserved in relation to other vertebrate and crustacean COXs. Expression of PmCOX in 293T cells increased levels of secreted PGE2 and PGF2α up to 60- and 77-fold, respectively, compared to control cells. Incubation of purified PmCOX with endoglycosidase H, which cleaves oligosaccharides from N-linked glycoproteins, reduced the molecular mass of PmCOX. Similarly, addition of tunicamycin, which inhibits N-linked glycosylation, in PmCOX-expressing cells resulted in PmCOX protein with lower molecular mass than those obtained from untreated cells, suggesting that PmCOX was N-glycosylated. Three potential glycosylation sites of PmCOX were identified at N79, N170 and N424. Mutational analysis revealed that although all three residues were glycosylated, only mutations at N170 and N424 completely abolished catalytic function. Inhibition of COX activity by ibuprofen treatment also decreased the levels of PGE2 in shrimp haemolymph. This study not only establishes the presence of the COX enzyme in penaeid shrimp, but also reveals that N-glycosylation sites are highly conserved and required for COX function in crustaceans.

scholarly journals Cleavage of Rubber by the Latex Clearing Protein (Lcp) of Streptomyces sp. Strain K30: Molecular Insights

2016 ◽  
Vol 82 (22) ◽  
pp. 6593-6602 ◽  
Author(s):  
Wolf Röther ◽  
Stefanie Austen ◽  
Jakob Birke ◽  
Dieter Jendrossek
Keyword(s):  
Biochemical Characterization ◽  
Mutational Analysis ◽  
Axial Position ◽  
Amino Acid Residues ◽  
Content Type ◽  
Research Activities ◽  
Domain Of Unknown Function ◽  
Arginine Residues ◽  
Aldehyde Groups ◽  
Key Residues

ABSTRACTGram-positive rubber degraders such asStreptomycessp. strain K30 cleave rubber [poly(cis-1,4-isoprene)] to low-molecular-mass oligoisoprenoid products with terminal keto and aldehyde groups by the secretion of a latex clearing protein (Lcp) designated rubber oxygenase. LcpK30is a hemebcytochrome and has a domain of unknown function (DUF2236) that is characteristic of orthologous Lcps. Proteins with a DUF2236 domain are characterized by three highly conserved residues (R164, T168, and H198 in LcpK30). Exchange of R164 or T168 by alanine and characterization of the purified LcpK30muteins revealed that both were stable and contained a heme group (red color) but were inactive. This finding identifies both residues as key residues for the cleavage reaction. The purified H198A mutein was also inactive and stable but was colorless due to the absence of heme. We constructed and characterized alanine muteins of four additional histidine residues moderately conserved in 495 LcpK30homologous sequences (H203A, H232A, H259A, H266A). All muteins revealed wild-type properties, excluding any importance for activity and/or heme coordination. Since LcpK30has only eight histidines and the three remaining residues (H103, H184, and H296) were not conserved (<11%), H198 presumably is the only essential histidine, indicating its putative function as a heme ligand. The second axial position of the heme is likely occupied by a not yet identified molecule. Mutational analysis of three strictly conserved arginine residues (R195, R202, R328) showed that R195A and R202A muteins were colorless and instable, suggesting that these residues are important for the protein stability.IMPORTANCELarge amounts of rubber waste materials have been permanently released into the environment for more than a century, yet accumulation of rubber particles released, e.g., by abrasion of tires along highways has not been observed. This is indicative of the ubiquitous presence and activity of rubber-degrading microorganisms. Despite increasing research activities on rubber biodegradation during the last 2 decades, the knowledge of the enzymatic cleavage mechanism of rubber by latex clearing protein (Lcp) still is limited. In particular, the catalytic cleavage mechanism and the amino acids of Lcp proteins (Lcps) that are involved have not yet been identified for any Lcp. In this study, we investigated the importance of 10 amino acid residues of Lcp fromStreptomycessp. K30 (LcpK30) by mutagenesis, mutein purification, and biochemical characterization. We identified several essential residues, one of which most likely represents an axial heme ligand in Lcp ofStreptomycessp. K30.

scholarly journals Biochemical and structural analyses of a bacterial endo-β-1,2-glucanase reveal a new glycoside hydrolase family

2017 ◽  
Vol 292 (18) ◽  
pp. 7487-7506 ◽  
Author(s):  
Koichi Abe ◽  
Masahiro Nakajima ◽  
Tetsuro Yamashita ◽  
Hiroki Matsunaga ◽  
Shinji Kamisuki ◽  
...  
Keyword(s):  
Active Site ◽  
Biochemical Characterization ◽  
Mutational Analysis ◽  
Sequence Similarity ◽  
Glycoside Hydrolases ◽  
Glycoside Hydrolase Family ◽  
Significant Sequence Similarity ◽  
Cell Extracts ◽  
Ligand Free ◽  
Complex Forms

β-1,2-Glucan is an extracellular cyclic or linear polysaccharide from Gram-negative bacteria, with important roles in infection and symbiosis. Despite β-1,2-glucan's importance in bacterial persistence and pathogenesis, only a few reports exist on enzymes acting on both cyclic and linear β-1,2-glucan. To this end, we purified an endo-β-1,2-glucanase to homogeneity from cell extracts of the environmental species Chitinophaga arvensicola, and an endo-β-1,2-glucanase candidate gene (Cpin_6279) was cloned from the related species Chitinophaga pinensis. The Cpin_6279 protein specifically hydrolyzed linear β-1,2-glucan with polymerization degrees of ≥5 and a cyclic counterpart, indicating that Cpin_6279 is an endo-β-1,2-glucananase. Stereochemical analysis demonstrated that the Cpin_6279-catalyzed reaction proceeds via an inverting mechanism. Cpin_6279 exhibited no significant sequence similarity with known glycoside hydrolases (GHs), and thus the enzyme defines a novel GH family, GH144. The crystal structures of the ligand-free and complex forms of Cpin_6279 with glucose (Glc) and sophorotriose (Glc-β-1,2-Glc-β-1,2-Glc) determined up to 1.7 Å revealed that it has a large cavity appropriate for polysaccharide degradation and adopts an (α/α)6-fold slightly similar to that of GH family 15 and 8 enzymes. Mutational analysis indicated that some of the highly conserved acidic residues in the active site are important for catalysis, and the Cpin_6279 active-site architecture provided insights into the substrate recognition by the enzyme. The biochemical characterization and crystal structure of this novel GH may enable discovery of other β-1,2-glucanases and represent a critical advance toward elucidating structure-function relationships of GH enzymes.

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