Phylogenetic, Molecular, and Biochemical Characterization of Caffeic Acid o-Methyltransferase Gene Family in Brachypodium distachyon

Caffeic acid o-methyltransferase (COMT) is one of the important enzymes controlling lignin monomer production in plant cell wall synthesis. Analysis of the genome sequence of the new grass model Brachypodium distachyon identified four COMT gene homologs, designated as BdCOMT1, BdCOMT2, BdCOMT3, and BdCOMT4. Phylogenetic analysis suggested that they belong to the COMT gene family, whereas syntenic analysis through comparisons with rice and sorghum revealed that BdCOMT4 on Chromosome 3 is the orthologous copy of the COMT genes well characterized in other grass species. The other three COMT genes are unique to Brachypodium since orthologous copies are not found in the collinear regions of rice and sorghum genomes. Expression studies indicated that all four Brachypodium COMT genes are transcribed but with distinct patterns of tissue specificity. Full-length cDNAs were cloned in frame into the pQE-T7 expression vector for the purification of recombinant Brachypodium COMT proteins. Biochemical characterization of enzyme activity and substrate specificity showed that BdCOMT4 has significant effect on a broad range of substrates with the highest preference for caffeic acid. The other three COMTs had low or no effect on these substrates, suggesting that a diversified evolution occurred on these duplicate genes that not only impacted their pattern of expression, but also altered their biochemical properties.

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Genome-wide Characterization of Major Intrinsic Protein (MIP) Gene Family in Brachypodium distachyon

Current Bioinformatics ◽

10.2174/1574893612666171023152558 ◽

2018 ◽

Vol 13 (5) ◽

pp. 536-552 ◽

Cited By ~ 4

Author(s):

Ankush Ashok Saddhe ◽

Shweta ◽

Kareem A. Mosa ◽

Kundan Kumar ◽

Manoj Prasad ◽

...

Keyword(s):

Gene Family ◽

Brachypodium Distachyon ◽

Major Intrinsic Protein ◽

Intrinsic Protein ◽

Genome Wide

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Biochemical characterization of specific Alanine Decarboxylase (AlaDC) and its ancestral enzyme Serine Decarboxylase (SDC) in tea plants (Camellia sinensis)

BMC Biotechnology ◽

10.1186/s12896-021-00674-x ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Peixian Bai ◽

Liyuan Wang ◽

Kang Wei ◽

Li Ruan ◽

Liyun Wu ◽

...

Keyword(s):

Gene Duplication ◽

Camellia Sinensis ◽

Biochemical Characterization ◽

Specific Activity ◽

Protein Sequences ◽

Biochemical Properties ◽

High Similarity ◽

New Gene ◽

Tea Plants

Abstract Background Alanine decarboxylase (AlaDC), specifically present in tea plants, is crucial for theanine biosynthesis. Serine decarboxylase (SDC), found in many plants, is a protein most closely related to AlaDC. To investigate whether the new gene AlaDC originate from gene SDC and to determine the biochemical properties of the two proteins from Camellia sinensis, the sequences of CsAlaDC and CsSDC were analyzed and the two proteins were over-expressed, purified, and characterized. Results The results showed that exon-intron structures of AlaDC and SDC were quite similar and the protein sequences, encoded by the two genes, shared a high similarity of 85.1%, revealing that new gene AlaDC originated from SDC by gene duplication. CsAlaDC and CsSDC catalyzed the decarboxylation of alanine and serine, respectively. CsAlaDC and CsSDC exhibited the optimal activities at 45 °C (pH 8.0) and 40 °C (pH 7.0), respectively. CsAlaDC was stable under 30 °C (pH 7.0) and CsSDC was stable under 40 °C (pH 6.0–8.0). The activities of the two enzymes were greatly enhanced by the presence of pyridoxal-5′-phosphate. The specific activity of CsSDC (30,488 IU/mg) was 8.8-fold higher than that of CsAlaDC (3467 IU/mg). Conclusions Comparing to CsAlaDC, its ancestral enzyme CsSDC exhibited a higher specific activity and a better thermal and pH stability, indicating that CsSDC acquired the optimized function after a longer evolutionary period. The biochemical properties of CsAlaDC might offer reference for theanine industrial production.

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Functional and Biochemical Characterization of Immunologically Derived Equine Platelet-Activating Factor

Veterinary Pathology ◽

10.1177/030098588502200413 ◽

1985 ◽

Vol 22 (4) ◽

pp. 375-386 ◽

Cited By ~ 6

Author(s):

H. C. Wimberly ◽

D. O. Slauson ◽

N. R. Neilsen

Keyword(s):

Functional Activity ◽

Biochemical Characterization ◽

Platelet Activating Factor ◽

Biochemical Properties ◽

Naturally Occurring ◽

Rabbit Platelets ◽

Rapid Reaction ◽

Specific Challenge

Antigen-specific challenge of equine leukocytes induced the non-lytic release of a platelet-activating factor in vitro. The equine platelet-activating factor stimulated the release of serotonin from equine platelets in a dose-responsive manner, independent of the presence of cyclo-oxygenase pathway inhibitors such as indomethacin. Rabbit platelets were also responsive to equine platelet-activating factor. The release of equine platelet-activating factor was a rapid reaction with near maximal secretion taking place in 30 seconds. Addition of equine platelet-activating factor to washed equine platelets stimulated platelet aggregation which could not be inhibited by the presence of aspirin or indomethacin. Platelets preincubated with equine platelet-activating factor became specifically desensitized to equine platelet-activating factor while remaining responsive to other platelet stimuli such as collagen and epinephrine. The following biochemical properties of equine platelet-activating factor are identical to those properties of 1-0-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC): stability upon exposure to air and acid; loss of functional activity after basecatalyzed methanolysis with subsequent acylation that returned all functional activity; and identical relative mobilities on silica gel G plates developed with chloroform:methanol:water (65:35:6, volume/volume). The combined functional and biochemical characteristics of equine platelet-activating factor strongly suggest identity between this naturally occurring, immunologically derived equine factor and AGEPC.

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Systematic Analysis and Biochemical Characterization of the Caffeoyl Shikimate Esterase Gene Family in Poplar

International Journal of Molecular Sciences ◽

10.3390/ijms222413366 ◽

2021 ◽

Vol 22 (24) ◽

pp. 13366

Author(s):

Xuechun Wang ◽

Nan Chao ◽

Aijing Zhang ◽

Jiaqi Kang ◽

Xiangning Jiang ◽

...

Keyword(s):

Gene Family ◽

Biochemical Characterization ◽

Phylogenetic Analyses ◽

Expression Patterns ◽

Lignin Biosynthesis ◽

Phenylpropanoid Pathway ◽

Biochemical Properties ◽

Phylogenetic Groups ◽

Systematic Analysis ◽

A Genome

Caffeoyl shikimate esterase (CSE) hydrolyzes caffeoyl shikimate into caffeate and shikimate in the phenylpropanoid pathway. In this study, we performed a systematic analysis of the CSE gene family and investigated the possible roles of CSE and CSE-like genes in Populus. We conducted a genome-wide analysis of the CSE gene family, including functional and phylogenetic analyses of CSE and CSE-like genes, using the poplar (Populus trichocarpa) genome. Eighteen CSE and CSE-like genes were identified in the Populus genome, and five phylogenetic groups were identified from phylogenetic analysis. CSEs in Group Ia, which were proposed as bona fide CSEs, have probably been lost in most monocots except Oryza sativa. Primary functional classification showed that PoptrCSE1 and PoptrCSE2 had putative function in lignin biosynthesis. In addition, PoptrCSE2, along with PoptrCSE12, might also respond to stress with a function in cell wall biosynthesis. Enzymatic assay of PoptoCSE1 (Populus tomentosa), -2 and -12 showed that PoptoCSE1 and -2 maintained CSE activity. PoptoCSE1 and 2 had similar biochemical properties, tissue expression patterns and subcellular localization. Most of the PoptrCSE-like genes are homologs of AtMAGL (monoacylglycerol lipase) genes in Arabidopsis and may function as MAG lipase in poplar. Our study provides a systematic understanding of this novel gene family and suggests the function of CSE in monolignol biosynthesis in Populus.

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Identification and biochemical characterization of a novel PP2C-like Ser/Thr phosphatase inE. coli

10.1101/303594 ◽

2018 ◽

Author(s):

Krithika Rajagopalan ◽

Jonathan Dworkin

Keyword(s):

Biochemical Characterization ◽

Regulatory Protein ◽

Biochemical Properties ◽

Bacterial Genomes ◽

Phosphatase Inhibitors ◽

E Coli ◽

Specificity And Sensitivity ◽

Genes Encoding ◽

Number Of Bacteria

AbstractIn bacteria, signaling phosphorylation is thought to occur primarily on His and Asp residues. However, phosphoproteomic surveys in phylogenetically diverse bacteria over the past decade have identified numerous proteins that are phosphorylated on Ser and/or Thr residues. Consistently, genes encoding Ser/Thr kinases are present in many bacterial genomes such asE. coli,which encodes at least three Ser/Thr kinases. Since Ser/Thr phosphorylation is a stable modification, a dedicated phosphatase is necessary to allow reversible regulation. Ser/Thr phosphatases belonging to several conserved families are found in bacteria. One family of particular interest are Ser/Thr phosphatases which have extensive sequence and structural homology to eukaryotic Ser/Thr PP2C phosphatases. These proteins, called eSTPs (eukaryotic-like Ser/Thr phosphatases), have been identified in a number of bacteria, but not inE. coli.Here, we describe a previously unknown eSTP encoded by anE. coliORF,yegK,and characterize its biochemical properties including its kinetics, substrate specificity and sensitivity to known phosphatase inhibitors. We investigate differences in the activity of this protein in closely relatedE. colistrains. Finally, we demonstrate that this eSTP acts to dephosphorylate a novel Ser/Thr kinase which is encoded in the same operon.ImportanceRegulatory protein phosphorylation is a conserved mechanism of signaling in all biological systems. Recent phosphoproteomic analyses of phylogenetically diverse bacteria including the model Gram-negative bacteriumE. colidemonstrate that many proteins are phosphorylated on serine or threonine residues. In contrast to phosphorylation on histidine or aspartate residues, phosphorylation of serine and threonine residues is stable and requires the action of a partner Ser/Thr phosphatase to remove the modification. Although a number of Ser/Thr kinases have been reported inE. coli, no partner Ser/Thrphosphatases have been identified. Here, we biochemically characterize a novel Ser/Thr phosphatase that acts to dephosphorylate a Ser/Thr kinase that is encoded in the same operon.

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Purification and biochemical characterization of a β-cyanoalanine synthase expressed in germinating seeds of Sorghum bicolor (L.) moench

Turkish Journal of Biochemistry ◽

10.1515/tjb-2017-0214 ◽

2018 ◽

Vol 43 (6) ◽

pp. 638-650

Author(s):

Ruth Ololade Amiola ◽

Adedeji Nelson Ademakinwa ◽

Zainab Adenike Ayinla ◽

Esther Nkechi Ezima ◽

Femi Kayode Agboola

Keyword(s):

Kinetic Parameters ◽

Biochemical Characterization ◽

Catalytic Properties ◽

Specific Activity ◽

Biochemical Properties ◽

Subunit Molecular Weight ◽

Cyanoalanine Synthase ◽

Germinating Seeds ◽

Sorghum Bicolor L

Abstract Background β-Cyanoalanine synthase plays essential roles in germinating seeds, such as in cyanide homeostasis. Methods β-Cyanoalanine synthase was isolated from sorghum seeds, purified using chromatographic techniques and its biochemical and catalytic properties were determined. Results The purified enzyme had a yield of 61.74% and specific activity of 577.50 nmol H2S/min/mg of protein. The apparent and subunit molecular weight for purified β-cyanoalanine synthase were 58.26±2.41 kDa and 63.4 kDa, respectively. The kinetic parameters with sodium cyanide as substrate were 0.67±0.08 mM, 17.60±0.50 nmol H2S/mL/min, 2.97×10−1 s−1 and 4.43×102 M−1 s−1 for KM, Vmax, kcat and kcat/KM, respectively. With L-cysteine as substrate, the kinetic parameters were 2.64±0.37 mM, 63.41±4.04 nmol H2S/mL/min, 10.71×10−1 s−1 and 4.06×102 M−1 s−1 for KM, Vmax, kcat and kcat/KM, respectively. The optimum temperature and pH for activity were 35°C and 8.5, respectively. The enzyme retained more than half of its activity at 40°C. Inhibitors such as HgCl2, EDTA, glycine and iodoacetamide reduced enzyme activity. Conclusion The biochemical properties of β-cyanoalanine synthase in germinating sorghum seeds highlights its roles in maintaining cyanide homeostasis.

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Coding, Expression, Targeting, Import and Processing of Distinct Polyphenoloxidases in Tissues of Higher Plants

10.32747/1994.7613008.bard ◽

1994 ◽

Author(s):

John Steffens ◽

Eithan Harel ◽

Alfred Mayer

Keyword(s):

Gene Family ◽

Food Production ◽

Higher Plants ◽

Biochemical Properties ◽

Plant Tissues ◽

Chloroplast Metabolism ◽

Enzymic Browning ◽

Physiological And Biochemical ◽

Insight Into

Polyphenol oxidase (PPO) catalyzes the oxidation of phenols to quinones at the expense of O2. PPOs are ubiquitous in higer plants, and their role in oxidative browning of plant tissues causes large annual losses to food production. Despite the importance of PPOs to agriculture, the function(s) of PPOs in higher plants are not understood. Among other roles, PPOs have been proposed to participate in aspects of chloroplast metabolism, based on their occurrence in plastids and high Km for O2. Due to the ability of PPO to catalyze formation of highly reactive quinones, PPOs have also been proposed to be involved in a wide array of defensive interactions with insect, bacterial, and fungal pests. Physiological and biochemical studies of PPO have provided few answers to the major problems of PPO function, subcellular localization, and biochemical properties. This proposal achieved the following major objectives: cloning of PPO cDNAs in potato and tomato; characterization of the tomato PPO gene family; antisense downregulation of the tomato PPO gene family; and reduction in post-harvest enzymic browning of potato through expression of antisense PPO genes under the control of tuber-specific promoters. In addition, we established the lumenal localization of PPO, characterized and clarified the means by which PPOs are imported and processed by chloroplasts, and provided insight into the factors which control localization of PPOs. This proposal has thereby provided fundamental advances in the understanding of this enzyme and the control of its expression.

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Functional characterization of cinnamyl alcohol dehydrogenase and caffeic acid O-methyltransferase in Brachypodium distachyon

BMC Biotechnology ◽

10.1186/1472-6750-13-61 ◽

2013 ◽

Vol 13 (1) ◽

pp. 61 ◽

Cited By ~ 53

Author(s):

Gina M Trabucco ◽

Dominick A Matos ◽

Scott J Lee ◽

Aaron J Saathoff ◽

Henry D Priest ◽

...

Keyword(s):

Alcohol Dehydrogenase ◽

Caffeic Acid ◽

Brachypodium Distachyon ◽

Functional Characterization ◽

Cinnamyl Alcohol Dehydrogenase ◽

Cinnamyl Alcohol

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Biochemical Characterization of a Bifunctional Enzyme Constructed by the Fusion of a Glucuronan Lyase and a Chitinase from Trichoderma sp.

Life ◽

10.3390/life10100234 ◽

2020 ◽

Vol 10 (10) ◽

pp. 234

Author(s):

Zeineb Baklouti ◽

Cédric Delattre ◽

Guillaume Pierre ◽

Christine Gardarin ◽

Slim Abdelkafi ◽

...

Keyword(s):

Biochemical Characterization ◽

Biochemical Properties ◽

Cell Wall Degrading Enzymes ◽

Promising Candidate ◽

Structural Modifications ◽

Chimeric Enzymes ◽

Bifunctional Enzymes ◽

Trichoderma Sp ◽

Ph Variations

Bifunctional enzymes created by the fusion of a glucuronan lyase (TrGL) and a chitinase (ThCHIT42) from Trichoderma sp. have been constructed with the aim to validate a proof of concept regarding the potential of the chimera lyase/hydrolase by analyzing the functionality and the efficiency of the chimeric constructions compared to parental enzymes. All the chimeric enzymes, including or nor linker (GGGGS), were shown functional with activities equivalent or higher to native enzymes. The velocity of glucuronan lyase was considerably increased for chimeras, and may involved structural modifications at the active site. The fusion has induced a slightly decrease of the thermostability of glucuronan lyase, without modifying its catalytic activity regarding pH variations ranging from 5 to 8. The biochemical properties of chitinase seemed to be more disparate between the different fusion constructions suggesting an impact of the linkers or structural interactions with the linked glucuronan lyase. The chimeric enzymes displayed a decreased stability to temperature and pH variations, compared to parental one. Overall, TrGL-ThCHIT42 offered the better compromise in terms of biochemical stability and enhanced activity, and could be a promising candidate for further experiments in the field of fungi Cell Wall-Degrading Enzymes (CWDEs).

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Genome-Wide Identification and Characterization of the SHI-Related Sequence Gene Family in Rice

Evolutionary Bioinformatics ◽

10.1177/1176934320941495 ◽

2020 ◽

Vol 16 ◽

pp. 117693432094149

Author(s):

Jun Yang ◽

Peng Xu ◽

Diqiu Yu

Keyword(s):

Oryza Sativa ◽

Gene Family ◽

Brachypodium Distachyon ◽

Functional Divergence ◽

Japonica Rice ◽

Segmental Duplications ◽

Related Sequence ◽

Genome Wide ◽

A Genome

Rice ( Oryza sativa) yield is correlated to various factors. Transcription regulators are important factors, such as the typical SHORT INTERNODES-related sequences (SRSs), which encode proteins with single zinc finger motifs. Nevertheless, knowledge regarding the evolutionary and functional characteristics of the SRS gene family members in rice is insufficient. Therefore, we performed a genome-wide screening and characterization of the OsSRS gene family in Oryza sativa japonica rice. We also examined the SRS proteins from 11 rice sub-species, consisting of 3 cultivars, 6 wild varieties, and 2 other genome types. SRS members from maize, sorghum, Brachypodium distachyon, and Arabidopsis were also investigated. All these SRS proteins exhibited species-specific characteristics, as well as monocot- and dicot-specific characteristics, as assessed by phylogenetic analysis, which was further validated by gene structure and motif analyses. Genome comparisons revealed that segmental duplications may have played significant roles in the recombination of the OsSRS gene family and their expression levels. The family was mainly subjected to purifying selective pressure. In addition, the expression data demonstrated the distinct responses of OsSRS genes to various abiotic stresses and hormonal treatments, indicating their functional divergence. Our study provides a good reference for elucidating the functions of SRS genes in rice.

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