scholarly journals Identification and Biochemical Characterization of Serine Hydroxymethyl Transferase in the Hydrogenosome of Trichomonas vaginalis

2006 ◽  
Vol 5 (12) ◽  
pp. 2072-2078 ◽  
Author(s):  
Mandira Mukherjee ◽  
Stuart A. Sievers ◽  
Mark T. Brown ◽  
Patricia J. Johnson
Keyword(s):  
Amino Acid ◽  
Active Site ◽  
Trichomonas Vaginalis ◽  
Pyridoxal Phosphate ◽  
Biochemical Characterization ◽  
Single Gene ◽  
Mammalian Host ◽  
Gene Encoding ◽  
Conserved Sequence ◽  
Serine Hydroxymethyl Transferase

ABSTRACT Serine hydroxymethyl transferase (SHMT) is a pyridoxal phosphate (PLP)-dependent enzyme that catalyzes the reversible conversion of serine and tetrahydrofolate to glycine and methylenetetrahydrofolate. We have identified a single gene encoding SHMT in the genome of Trichomonas vaginalis, an amitochondriate, deep-branching unicellular protist. The protein possesses a putative N-terminal hydrogenosomal presequence and was shown to localize to hydrogensomes by immunofluorescence analysis, providing evidence of amino acid metabolism in this unusual organelle. In contrast to the tetrameric SHMT that exists in the mammalian host, we found that the T. vaginalis SHMT is a homodimer, as found in prokaryotes. All examined SHMT contain an 8-amino-acid conserved sequence, VTTTTHKT, containing the active-site lysyl residue (Lys 251 in TvSHMT) that forms an internal aldimine with PLP. We mutated this Lys residue to Arg and Gln and examined structural and catalytic properties of the wild-type and mutant enzymes in comparison to that reported for the mammalian protein. The oligomeric structure of the mutant K251R and K251Q TvSHMT was not affected, in contrast to that observed for comparable mutations in the mammalian enzyme. Likewise, contrary to that observed for mammalian SHMT, the catalytic activity of K251R TvSHMT was unaffected in the presence of PLP. The K251Q TvSHMT, however, was found to be inactive. These studies indicate that the active site of the parasite enzyme is distinct from its prokaryotic and eukaryotic counterparts and identify TvSHMT as a potential drug target.

scholarly journals The mechanism of action of serine transhydroxymethylase

1970 ◽  
Vol 116 (2) ◽  
pp. 277-286 ◽  
Author(s):  
P. M. Jordan ◽  
M. Akhtar
Keyword(s):  
Schiff Base ◽  
Amino Acid ◽  
Hydrogen Atom ◽  
Mechanism Of Action ◽  
Active Site ◽  
Enzyme Preparation ◽  
Pyridoxal Phosphate ◽  
Acid Oxidase ◽  
Amino Acid Oxidase

1. The preparation of stereospecifically tritiated glycines and the determination of their absolute configurations by the use of d-amino acid oxidase are described. 2. The reaction catalysed by serine transhydroxymethylase, which results in the conversion of glycine into serine, has been separated into at least four partial reactions. It is suggested that the first event in this conversion is the formation of a Schiff base intermediate of glycine and pyridoxal phosphate. The next important step involves the removal of the 2S-hydrogen atom of glycine to give a carbanion intermediate. Experiments pertinent to the mechanism of conversion of this carbanion intermediate into serine are described. 3. The enzyme preparation catalysing the conversion of glycine into serine also participates in the conversion of glycine into threonine and allothreonine. In both these conversions, glycine → serine and glycine → threonine, the 2S-hydrogen atom of glycine is eliminated and the 2R-hydrogen atom of glycine is retained. 4. In the light of these experiments the mechanism of action of serine transhydroxymethylase is discussed. It is suggested that methylenetetrahydrofolate is the carrier of formaldehyde, from which formaldehyde may be liberated at the active site of the enzyme, thus allowing the overall reaction to take place.

scholarly journals Cloning, expression, and biochemical characterization of a cold-active GDSL-esterase of a Pseudomonas sp. S9 isolated from Spitsbergen island soil.

2016 ◽  
Vol 63 (1) ◽  
Author(s):  
Monika Wicka ◽  
Marta Wanarska ◽  
Ewelina Krajewska ◽  
Anna Pawlak-Szukalska ◽  
Józef Kur ◽  
...  
Keyword(s):  
Amino Acid ◽  
Biochemical Characterization ◽  
Catalytic Domain ◽  
Pseudomonas Sp ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
Optimal Activity ◽  
Cold Active ◽  
Psychrotolerant Bacterium ◽  
Type V

An estS9 gene, encoding an esterase of the psychrotolerant bacterium Pseudomonas sp. S9 was cloned and sequenced. The deduced sequence revealed a protein of 636 amino acid residues with a molecular mass of 69 kDa. Further amino acid sequence analysis revealed that the EstS9 enzyme contained a G-D-S-L motif centered at a catalytic serine, an N-terminal catalytic domain and a C-terminal autotransporter domain. Two recombinant E. coli strains for production of EstS9N (a two domain enzyme) and EstS9Δ (a one domain enzyme) proteins were constructed, respectively. Both recombinant proteins were successfully produced as inclusion bodies and then purified under denaturing conditions. However, because of the low enzymatic activity of the refolded EstS9Δ protein, only the EstS9N protein was further characterized. The purified and refolded EstS9N protein was active towards short-chain p-nitrophenyl esters (C2-C8), with optimal activity for the butyrate (C4) ester. With p-nitrophenyl butyrate as the substrate, the enzyme displayed optimal activity at 35°C and pH 9.0. Additionally, the EstS9N esterase retained ~90% of its activity from 25-40°C and ~40% of its activity at 10°C. Moreover, analysis of its kinetic parameters (Km, kcat, kcat/Km) toward p-nitrophenyl butyrate determined at 15°C and 25°C confirmed that the EstS9 enzyme is cold-adapted. To the best of our knowledge, EstS9 is the third characterized cold-active GDSL-esterase and the first one confirmed to contain an autotransporter domain characteristic for enzymes secreted by the type V secretion system.

scholarly journals Molecular cloning, expression and characterization of pyridoxamine–pyruvate aminotransferase

2006 ◽  
Vol 396 (3) ◽  
pp. 499-507 ◽  
Author(s):  
Yu Yoshikane ◽  
Nana Yokochi ◽  
Kouhei Ohnishi ◽  
Hideyuki Hayashi ◽  
Toshiharu Yagi
Keyword(s):  
Amino Acid ◽  
Active Site ◽  
Lysine Residue ◽  
Type I ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
Mesorhizobium Loti ◽  
Fold Type ◽  
Km Value ◽  
Schiff Base Formation

Pyridoxamine–pyruvate aminotransferase is a PLP (pyridoxal 5′-phosphate) (a coenzyme form of vitamin B6)-independent aminotransferase which catalyses a reversible transamination reaction between pyridoxamine and pyruvate to form pyridoxal and L-alanine. The gene encoding the enzyme has been identified, cloned and overexpressed for the first time. The mlr6806 gene on the chromosome of a symbiotic nitrogen-fixing bacterium, Mesorhizobium loti, encoded the enzyme, which consists of 393 amino acid residues. The primary sequence was identical with those of archaeal aspartate aminotransferase and rat serine–pyruvate aminotransferase, which are PLP-dependent aminotransferases. The results of fold-type analysis and the consensus amino acid residues found around the active-site lysine residue identified in the present study showed that the enzyme could be classified into class V aminotransferases of fold type I or the AT IV subfamily of the α family of the PLP-dependent enzymes. Analyses of the absorption and CD spectra of the wild-type and point-mutated enzymes showed that Lys197 was essential for the enzyme activity, and was the active-site lysine residue that corresponded to that found in the PLP-dependent aminotransferases, as had been suggested previously [Hodsdon, Kolb, Snell and Cole (1978) Biochem. J. 169, 429–432]. The Kd value for pyridoxal determined by means of CD was 100-fold lower than the Km value for it, suggesting that Schiff base formation between pyridoxal and the active-site lysine residue is partially rate determining in the catalysis of pyridoxal. The active-site structure and evolutionary aspects of the enzyme are discussed.

scholarly journals Molecular cloning and characterization of the structural gene for the major iron-regulated protein expressed by Neisseria gonorrhoeae.

1990 ◽  
Vol 171 (5) ◽  
pp. 1535-1546 ◽  
Author(s):  
S A Berish ◽  
T A Mietzner ◽  
L W Mayer ◽  
C A Genco ◽  
B P Holloway ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Neisseria Gonorrhoeae ◽  
Biochemical Characterization ◽  
Iron Acquisition ◽  
Regulatory Region ◽  
Single Copy ◽  
Oligonucleotide Probes ◽  
Gene Encoding

This report describes the cloning and sequencing of the major iron-regulated protein (termed Fbp) of Neisseria gonorrhoeae strain F62. Attempts to identify recombinants expressing the Fbp using specific antibody proved unsuccessful. Therefore, an alternative cloning strategy using oligonucleotide probes derived from NH2-terminal and tryptic fragments of this protein was used to identify short fragments of the gene. Using this methodology, the gene encoding the precursor of Fbp was cloned on three separate overlapping fragments and sequenced, and the amino acid sequence was deduced. These data were unambiguously confirmed by the known NH2-terminal amino acid sequence and were supported by the sequences from tryptic fragments that lie outside of this region. Using oligonucleotide probes, we were unable to obtain clones encoding the potential regulatory region of this protein. Therefore, the technique of inverse polymerase chain reaction was used to amplify a fragment containing an additional 200 bp. This fragment was cloned and sequenced and found to contain a consensus ribosome binding site and potential -10 and -35 sequences. Hybridization analysis of genomic DNA from gonococcal strain F62 indicated that only a single copy of the Fbp gene exists per genome. These results complement the biochemical characterization of the Fbp expressed by gonococci and further suggest that it has a role in iron-acquisition.

Use of inhibitors to study reactions catalyzed by enzymes requiring pyridoxal phosphate as coenzyme

2000 ◽  
Vol 72 (3) ◽  
pp. 373-384 ◽  
Author(s):  
Benjamin Adams ◽  
B. Svante Axelsson ◽  
Kenneth J. M. Beresford ◽  
Nicola J. Church ◽  
Philip A. Spencer ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Active Site ◽  
Aspartate Aminotransferase ◽  
Pyridoxal Phosphate ◽  
Family Formation ◽  
Enzyme Inhibitors ◽  
Acid Oxidase ◽  
Amino Acid Oxidase ◽  
Naturally Occurring

The stereochemistry of a variety of pyridoxal phosphate-mediated enzymic reactions has been studied using enzyme inhibitors that are stereospecifically labeled in the β-position with deuterium. A versatile synthesis has been developed to prepare a wide variety of stereospecifically labeled d- and l-amino acids and inhibitors. Investigation of the "turnover" of β-chloro-d-alanine and d- and l-serine-O-sulfate by d-amino acid aminotransferase and l-aspartate aminotransferase respectively has shown that reaction within the active site of the former enzyme occurs with retention of stereochemistry. Although l-aspartate aminotransferase is an enzyme of the α-family, when it was incubated with β-chloro-l-alanine in the presence of 2-mercaptoethanol, β-substitution occurred. This was shown to involve retention of stereochemistry, an outcome typical of reactions catalyzed by enzymes of the β-family that have little or no homology with enzymes of the α-family. Formation of the "Schnackerz intermediate" has been studied as has the d-amino acid oxidase catalyzed reaction of the naturally occurring inhibitor d-propargylglycine.

scholarly journals Inducible expression of calmodulin antisense RNA in Dictyostelium cells inhibits the completion of cytokinesis.

1992 ◽  
Vol 3 (12) ◽  
pp. 1403-1413 ◽  
Author(s):  
T Liu ◽  
J G Williams ◽  
M Clarke
Keyword(s):  
Amino Acid ◽  
Cell Density ◽  
Single Gene ◽  
Gene Encoding ◽  
Protein Levels ◽  
Multinucleated Cells ◽  
Cell Cleavage ◽  
Base Pair Fragment ◽  
Cytoplasmic Bridges ◽  
High Level

The single gene encoding calmodulin in the eukaryotic microorganism Dictyostelium discoideum was cloned and sequenced. The gene was found to contain three introns, one lying immediately after the translation initiation codon. The deduced amino acid sequence indicated that Dictyostelium calmodulin contains 19 amino acid differences from vertebrate calmodulin, including extensions at both termini. Northern blot analysis showed that similar levels of calmodulin mRNA are present throughout growth and development of wild-type cells. A complete copy of the calmodulin cDNA was prepared, and an 87-base pair fragment complementary to the 5'-end of the calmodulin mRNA was subcloned into the Dictyostelium transformation vector pVEII, such that expression of the antisense transcript was driven by the discoidin I gamma promoter. Transformed cells were selected and maintained at low cell density, a condition resulting in minimal activity of the discoidin I promoter. High level expression was induced by allowing the transformants to reach high cell density or by growing them in the presence of medium conditioned by high density cells. Under these conditions, in which calmodulin mRNA and protein levels were reduced about twofold, the calmodulin antisense transformants lost the ability to complete cytokinesis. A contractile ring formed and constricted, but the midbody linking daughter cells failed to break. The resulting cell population contained multinucleated cells and networks of cells connected by cytoplasmic bridges. Normal cell division was restored when the cells were diluted to low density. These observations have identified a new point at which calmodulin may regulate cell cleavage.

scholarly journals Genetic and Biochemical Characterization of a Highly Thermostable α-l-Arabinofuranosidase fromThermobacillus xylanilyticus

2000 ◽  
Vol 66 (4) ◽  
pp. 1734-1736 ◽  
Author(s):  
Takoua Debeche ◽  
Nicola Cummings ◽  
Ian Connerton ◽  
Philippe Debeire ◽  
Michael J. O'Donohue
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Classification System ◽  
Biochemical Characterization ◽  
Glycosyl Hydrolase ◽  
Gene Encoding ◽  
Ph Range ◽  
Sequence Similarities

ABSTRACT The gene encoding an α-l-arabinofuranosidase fromThermobacillus xylanilyticus D3, AbfD3, was isolated. Characterization of the purified recombinant α-l-arabinofuranosidase produced in Escherichia coli revealed that it is highly stable with respect to both temperature (up to 90°C) and pH (stable in the pH range 4 to 12). On the basis of amino acid sequence similarities, this 56,071-Da enzyme could be assigned to family 51 of the glycosyl hydrolase classification system. However, substrate specificity analysis revealed that AbfD3, unlike the majority of F51 members, displays high activity in the presence of polysaccharides.

scholarly journals Novel Maltogenic Amylase CoMA fromCorallococcussp. Strain EGB Catalyzes the Conversion of Maltooligosaccharides and Soluble Starch to Maltose

2018 ◽  
Vol 84 (14) ◽  
Author(s):  
Jie Zhou ◽  
Zhoukun Li ◽  
Han Zhang ◽  
Jiale Wu ◽  
Xianfeng Ye ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Biochemical Characterization ◽  
Soluble Starch ◽  
Glycoside Hydrolase Family ◽  
Gene Encoding ◽  
Commercial Development ◽  
Amylolytic Enzyme ◽  
Content Type ◽  
High Level

ABSTRACTThe gene encoding the novel amylolytic enzyme designated CoMA was cloned fromCorallococcussp. strain EGB. The deduced amino acid sequence contained a predicted lipoprotein signal peptide (residues 1 to 18) and a conserved glycoside hydrolase family 13 (GH13) module. The amino acid sequence of CoMA exhibits low sequence identity (10 to 19%) with cyclodextrin-hydrolyzing enzymes (GH13_20) and is assigned to GH13_36. The most outstanding feature of CoMA is its ability to catalyze the conversion of maltooligosaccharides (≥G3) and soluble starch to maltose as the sole hydrolysate. Moreover, it can hydrolyze γ-cyclodextrin and starch to maltose and hydrolyze pullulan exclusively to panose with relative activities of 0.2, 1, and 0.14, respectively. CoMA showed both hydrolysis and transglycosylation activities toward α-1,4-glycosidic bonds but not to α-1,6-linkages. Moreover, glucosyl transfer was postulated to be the major transglycosidation reaction for producing a high level of maltose without the attendant production of glucose. These results indicated that CoMA possesses some unusual properties that distinguish it from maltogenic amylases and typical α-amylases. Its physicochemical properties suggested that it has potential for commercial development.IMPORTANCEThe α-amylase fromCorallococcussp. EGB, which was classified to the GH13_36 subfamily, can catalyze the conversion of maltooligosaccharides (≥G3) and soluble starch to maltose as the sole hydrolysate. An action mechanism for producing a high level of maltose without the attendant production of glucose has been proposed. Moreover, it also can hydrolyze γ-cyclodextrin and pullulan. Its biochemical characterization suggested that CoMA may be involved the accumulation of maltose inCorallococcusmedia.

Identification and Bioinformatics Analysis of Cysteine Synthase from the Filamentous Fungus, Monascus purpureus

2014 ◽  
Vol 522-524 ◽  
pp. 272-275
Author(s):  
Nan Qing Liao ◽  
Jiang Ning Yao ◽  
Hao Ming Li
Keyword(s):  
Amino Acid ◽  
Pyridoxal Phosphate ◽  
Structural Model ◽  
Bioinformatics Analysis ◽  
Tertiary Structure ◽  
Monascus Purpureus ◽  
Gene Encoding ◽  
Cysteine Synthase ◽  
Comparison Results ◽  
Alignment Analysis

A gene encoding a putative cysteine synthase was obtained by screening Monascus purpureus cDNA library. Bioinformatics analysis showed that this protein has Rhodanese Homology Domain in C-terminal, and Pyridoxal-phosphate dependent enzyme domain in N-terminal, and CBS-like structure. The deduced cysteine synthase protein of M. purpureus contained 517 amino acid, with molecular mass of 57,044Da. Sequence alignment analysis revealed that M. purpureus deduced cysteine synthase was closely related to cysteine synthase from Aspergillus, Ajellomyces and Paracoccidioides, and highly homologous to aforementioned and other known cysteine synthase. The structural model of the deduced cysteine synthase closely match the template with 100% confidence and 20-30% identity. The consistency of the comparison results of the primary structure, secondary structure and tertiary structure suggests that the dedued protein may well be cysteine synthase.

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