Molecular and biochemical characterization of Paragonimus westermani tyrosinase

Parasitology ◽  
2015 ◽  
Vol 142 (6) ◽  
pp. 807-815 ◽  
Author(s):  
Y.-A. BAE ◽  
S.-H. KIM ◽  
C.-S. AHN ◽  
J.-G. KIM ◽  
Y. KONG
Keyword(s):  
Oxidase Activity ◽  
Biochemical Characterization ◽  
Hydroxyl Groups ◽  
Reading Frame ◽  
Paragonimus Westermani ◽  
Rate Limiting Step ◽  
Tanning Process ◽  
Weak Activity ◽  
Rate Limiting

SUMMARYTrematode tyrosinases (TYRs) play a major role in the tanning process during eggshell formation. We investigated the molecular and biochemical features of Paragonimus westermani TYR (PwTYR). The PwTYR cDNA was composed of 1568-bp encompassing a 1422-bp-long open reading frame (474-amino acid polypeptide). A strong phylogenetic relationship with Platyhelminthes and Deuterostomian orthologues was evident. The recombinant PwTYR expressed in prokaryotic cells promptly oxidized diphenol substrates, with a preferential affinity toward ortho-positioned hydroxyl groups. It demonstrated fairly weak activity for monophenol compounds. Diphenol oxidase activity was augmented with an increase of pH from 5·0 to 8·0, while monophenol oxidase activity was highest at an acidic pH and gradually decreased as pH increased. Transcription profile of PwTYR was temporally upregulated along with worm development. PwTYR was specifically localized in vitellocytes and eggs. The results suggested that conversion of tyrosine to L-dihydroxyphenylalanine by PwTYR monophenol oxidase activity might be rate-limiting step during the sclerotization process of P. westermani eggs. The pH-dependent pattern of monophenol and diphenol oxidase activity further proposes that the initial hydroxylation might slowly but steadily progress in acidic secreted vesicles of vitellocytes and the second oxidation process might be rapidly accelerated by neural or weak alkaline pH environments within the ootype.

Heme Oxygenase-1/Carbon Monoxide: From Basic Science to Therapeutic Applications

2006 ◽  
Vol 86 (2) ◽  
pp. 583-650 ◽  
Author(s):  
Stefan W. Ryter ◽  
Jawed Alam ◽  
Augustine M. K. Choi
Keyword(s):  
Carbon Monoxide ◽  
Cellular Proliferation ◽  
Biochemical Characterization ◽  
Apoptotic Cell ◽  
Heme Oxygenase 1 ◽  
Bile Pigments ◽  
Cellular Functions ◽  
Rate Limiting Step ◽  
Rate Limiting

The heme oxygenases, which consist of constitutive and inducible isozymes (HO-1, HO-2), catalyze the rate-limiting step in the metabolic conversion of heme to the bile pigments (i.e., biliverdin and bilirubin) and thus constitute a major intracellular source of iron and carbon monoxide (CO). In recent years, endogenously produced CO has been shown to possess intriguing signaling properties affecting numerous critical cellular functions including but not limited to inflammation, cellular proliferation, and apoptotic cell death. The era of gaseous molecules in biomedical research and human diseases initiated with the discovery that the endothelial cell-derived relaxing factor was identical to the gaseous molecule nitric oxide (NO). The discovery that endogenously produced gaseous molecules such as NO and now CO can impart potent physiological and biological effector functions truly represented a paradigm shift and unraveled new avenues of intense investigations. This review covers the molecular and biochemical characterization of HOs, with a discussion on the mechanisms of signal transduction and gene regulation that mediate the induction of HO-1 by environmental stress. Furthermore, the current understanding of the functional significance of HO shall be discussed from the perspective of each of the metabolic by-products, with a special emphasis on CO. Finally, this presentation aspires to lay a foundation for potential future clinical applications of these systems.

scholarly journals Characterization of a novel N-acetylneuraminic acid lyase favoring industrial N-acetylneuraminic acid synthesis process

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Wenyan Ji ◽  
Wujin Sun ◽  
Jinmei Feng ◽  
Tianshun Song ◽  
Dalu Zhang ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Acid Synthesis ◽  
Synthesis Process ◽  
Acetylneuraminic Acid ◽  
Cleavage Activity ◽  
Rate Limiting Step ◽  
Synthesis Activity ◽  
Rate Limiting ◽  
Class I Aldolase

Abstract N-Acetylneuraminic acid lyase (NAL, E.C. number 4.1.3.3) is a Class I aldolase that catalyzes the reversible aldol cleavage of N-acetylneuraminic acid (Neu5Ac) from pyruvate and N-acetyl-D-mannosamine (ManNAc). Due to the high Neu5Ac cleavage activity in most isozyme forms, the enzyme catalyzes the rate-limiting step of two biocatalytic reactions producing Neu5Ac in industry. We report the biochemical characterization of a novel NAL from a “GRAS” (General recognized as safe) strain C. glutamicum ATCC 13032 (CgNal). Compared to all previously reported NALs, CgNal exhibited the lowest apparent k cat/Km value for Neu5Ac and highest apparent k cat/Km values for ManNAc and pyruvate, which makes CgNal favor industrial Neu5Ac synthesis process in a non-equilibrium condition. The recombinant CgNal reached the highest expression level (480 mg/L culture) and the highest reported yield of Neu5Ac was achieved (194 g/L, 0.63 M). All these unique properties make CgNal a promising biocatalyst for industrial Neu5Ac biosynthesis. Additionally, although showing the best Neu5Ac synthesis activity among the NAL family, CgNal is more related to dihydrodipicolinate synthase (DHDPS) by phylogenetic analysis. The activities of CgNal towards both NAL's and DHDPS' substrates are fairly high, which indicates CgNal a bi-functional enzyme. The sequence analysis suggests that CgNal might have adopted a unique set of residues for substrates recognition.

scholarly journals Biochemical Characterization of the Ran-RanBP1-RanGAP System: Are RanBP Proteins and the Acidic Tail of RanGAP Required for the Ran-RanGAP GTPase Reaction?

2003 ◽  
Vol 23 (22) ◽  
pp. 8124-8136 ◽  
Author(s):  
Michael J. Seewald ◽  
Astrid Kraemer ◽  
Marian Farkasovsky ◽  
Carolin Körner ◽  
Alfred Wittinghofer ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Interaction Analysis ◽  
Catalytic Efficiency ◽  
Diffusion Control ◽  
Microtubule Organization ◽  
Rate Limiting Step ◽  
Association Reaction ◽  
Rate Limiting ◽  
Fold Stimulation

ABSTRACT RanBP type proteins have been reported to increase the catalytic efficiency of the RanGAP-mediated GTPase reaction on Ran. Since the structure of the Ran-RanBP1-RanGAP complex showed RanBP1 to be located away from the active site, we reinvestigated the reaction using fluorescence spectroscopy under pre-steady-state conditions. We can show that RanBP1 indeed does not influence the rate-limiting step of the reaction, which is the cleavage of GTP and/or the release of product Pi. It does, however, influence the dynamics of the Ran-RanGAP interaction, its most dramatic effect being the 20-fold stimulation of the already very fast association reaction such that it is under diffusion control (4.5 × 108 M−1 s−1). Having established a valuable kinetic system for the interaction analysis, we also found, in contrast to previous findings, that the highly conserved acidic C-terminal end of RanGAP is not required for the switch-off reaction. Rather, genetic experiments in Saccharomyces cerevisiae demonstrate a profound effect of the acidic tail on microtubule organization during mitosis. We propose that the acidic tail of RanGAP is required for a process during mitosis.

scholarly journals Genetic Context and Biochemical Characterization of the IMP-18 Metallo-β-Lactamase Identified in aPseudomonas aeruginosaIsolate from the United States

2010 ◽  
Vol 55 (1) ◽  
pp. 140-145 ◽  
Author(s):  
Luisa Borgianni ◽  
Silvia Prandi ◽  
Laurie Salden ◽  
Gisela Santella ◽  
Nancy D. Hanson ◽  
...  
Keyword(s):  
United States ◽  
Biochemical Characterization ◽  
Gene Cassette ◽  
The United States ◽  
Turnover Rates ◽  
Reading Frame ◽  
Class 1 ◽  
Original Array ◽  
Genetic Context

ABSTRACTThe production of metallo-β-lactamase (MBL) is an important mechanism of resistance to β-lactam antibiotics, including carbapenems. Despite the discovery and emergence of many acquired metallo-β-lactamases, IMP-type determinants (now counting at least 27 variants) remain the most prevalent in some geographical areas. In Asian countries, and notably Japan, IMP-1 and its closely related variants are most widespread. Some other variants have been detected in other countries and show either an endemic (e.g., IMP-13 in Italy) or sporadic (e.g., IMP-12 in Italy or IMP-18 in the United States) occurrence. The IMP-18-producingPseudomonas aeruginosastrain PS 297 from the southwestern United States carried at least two class 1 integrons. One was identical to In51, while the other, named In133and carrying theblaIMP-18gene cassette in the third position, showed an original array of five gene cassettes, includingaacA7,qacF,aadA1, and an unknown open reading frame (ORF). Interestingly. In133differed significantly from In96, theblaIMP-18-carrying integron identified in aP. aeruginosaisolate from Mexico. The meropenem and ertapenem MIC values were much lower forEscherichia colistrains producing IMP-18 (0.06 and 0.12 μg/ml, respectively) than for strains producing IMP-1 (2 μg/ml for each). Kinetic data obtained with the purified enzyme revealed lower turnover rates of IMP-18 than of other IMP-type enzymes with most substrates.

scholarly journals Molecular and biochemical characterization of hemoglobinase, a cysteine proteinase, in Paragonimus westermani

2006 ◽  
Vol 44 (3) ◽  
pp. 187 ◽  
Author(s):  
Joon-Hyuck Choi ◽  
Jae-Hyuk Lee ◽  
Hak-Sun Yu ◽  
Hae-Jin Jeong ◽  
Jin Kim ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Cysteine Proteinase ◽  
Paragonimus Westermani

Wavelet Based Structural Analysis of Electroless Deposits in the Diffusion Limited Regime

1994 ◽  
Vol 367 ◽  
Author(s):  
A. Arneodo ◽  
F. Argoul ◽  
A. Kuhn ◽  
J.F. Muzy
Keyword(s):  
Electroless Deposition ◽  
Side Reactions ◽  
Statistical Features ◽  
Diffusion Limited ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Fractal Patterns ◽  
Rate Limiting ◽  
Comparative Structural Characterization

AbstractWe discuss the actual relevance of thin gap geometry electrodeposition to generate fractal patterns that mimic the morphology of Witten and Sander's diffusion-limited aggregates (DLA). Eliminating migration and convection, as well as electrochemical side reactions, we show that electroless deposition is a good candidate to meet the requirements for diffusion to be the rate limiting step of the growth process. We use the wavelet transform microscope to achieve a comparative structural characterization of both experimental electroless deposits and numerical DLA clusters. The fact that five-fold symmetry and Fibonacci hierarchical ordering are found as common predominant statistical features is, to our knowledge, the first demonstration, relying on an appropriate structural fractal analysis, of the existence of DLA morphologies in an experimental context.

scholarly journals Functional and biochemical characterization of a recombinant Arabidopsis thaliana 3-deoxy-D-manno-octulosonate 8-phosphate synthase

2004 ◽  
Vol 381 (1) ◽  
pp. 185-193 ◽  
Author(s):  
Jing WU ◽  
Mayur A. PATEL ◽  
Appavu K. SUNDARAM ◽  
Ronald W. WOODARD
Keyword(s):  
Arabidopsis Thaliana ◽  
Molecular Mass ◽  
Biochemical Characterization ◽  
Dipicolinic Acid ◽  
Kinetic Studies ◽  
Maximum Activity ◽  
Reading Frame ◽  
Sequential Mechanism ◽  
Phosphate Synthase

An open reading frame, encoding for KDOPS (3-deoxy-D-manno-octulosonate 8-phosphate synthase), from Arabidopsis thaliana was cloned into a T7-driven expression vector. The protein was overexpressed in Escherichia coli and purified to homogeneity. Recombinant A. thaliana KDOPS, in solution, displays an apparent molecular mass of 76 kDa and a subunit molecular mass of 31.519 kDa. Unlike previously studied bacterial KDOPSs, which are tetrameric, A. thaliana KDOPS appears to be a dimer in solution. The optimum temperature of the enzyme is 65 °C and the optimum pH is 7.5, with a broad peak between pH 6.5 and 9.5 showing 90% of maximum activity. The enzyme cannot be inactivated by EDTA or dipicolinic acid treatment, nor it can be activated by a series of bivalent metal ions, suggesting that it is a non-metallo-enzyme, as opposed to the initial prediction that it would be a metallo-enzyme. Kinetic studies showed that the enzyme follows a sequential mechanism with Km=3.6 μM for phosphoenolpyruvate and 3.8 μM for D-arabinose 5-phosphate and kcat=5.9 s−1 at 37 °C. On the basis of the characterization of A. thaliana KDOPS and phylogenetic analysis, plant KDOPSs may represent a new, distinct class of KDOPSs.

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