Molecular and biochemical characterization of herbicide-resistant mutants of cyanobacteria reveals that phytoene desaturation is a rate-limiting step in carotenoid biosynthesis

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.

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

Physiological Reviews ◽

10.1152/physrev.00011.2005 ◽

2006 ◽

Vol 86 (2) ◽

pp. 583-650 ◽

Cited By ~ 1481

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.

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

Scientific Reports ◽

10.1038/srep09341 ◽

2015 ◽

Vol 5 (1) ◽

Cited By ~ 12

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.

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Biochemical characterization of a trypanosome enzyme with glutathione-dependent peroxidase activity

Biochemical Journal ◽

10.1042/bj3520755 ◽

2000 ◽

Vol 352 (3) ◽

pp. 755-761 ◽

Cited By ~ 23

Author(s):

Shane R. WILKINSON ◽

David J. MEYER ◽

John M. KELLY

Keyword(s):

Linoleic Acid ◽

Peroxidase Activity ◽

Biochemical Characterization ◽

Linoleic Acid Hydroperoxide ◽

Butyl Hydroperoxide ◽

Rate Limiting Step ◽

Limiting Step ◽

Wide Range ◽

Acid Hydroperoxide ◽

Rate Limiting

In most eukaryotes, glutathione-dependent peroxidases play a key role in the metabolism of peroxides. Numerous studies have reported that trypanosomatids lack this activity. Here we show that this is not the case, at least for the American trypanosome Trypanosoma cruzi. We have isolated a single-copy gene from T. cruzi with the potential to encode an 18kDa enzyme, the sequence of which has highest similarity with glutathione peroxidases from plants. A recombinant form of the protein was purified following expression in Escherichia coli. The enzyme was shown to have peroxidase activity in the presence of glutathione/glutathione reductase but not in the presence of trypanothione/trypanothione reductase. It could metabolize a wide range of hydroperoxides (linoleic acid hydroperoxide and phosphatidylcholine hydroperoxide> cumene hydroperoxide>t-butyl hydroperoxide), but no activity towards hydrogen peroxide was detected. Enzyme activity could be saturated by glutathione when both fatty acid and short-chain organic hydroperoxides were used as substrate. For linoleic acid hydroperoxide, the rate-limiting step of this reaction is the reduction of the peroxidase by glutathione. With lower-affinity substrates such as t-butyl hydroperoxide, the rate-limiting step is the reduction of the oxidant. The data presented here identify a new arm of the T. cruzi oxidative defence system.

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Molecular and biochemical characterization of Paragonimus westermani tyrosinase

Parasitology ◽

10.1017/s0031182014001942 ◽

2015 ◽

Vol 142 (6) ◽

pp. 807-815 ◽

Cited By ~ 5

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.23.22.8124-8136.2003 ◽

2003 ◽

Vol 23 (22) ◽

pp. 8124-8136 ◽

Cited By ~ 35

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.

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