scholarly journals Novel Type of Glucose-6-Phosphate Isomerase in the Hyperthermophilic Archaeon Pyrococcus furiosus

2001 ◽  
Vol 183 (11) ◽  
pp. 3428-3435 ◽  
Author(s):  
Thomas Hansen ◽  
Margitta Oehlmann ◽  
Peter Schönheit
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Physiological Function ◽  
Pyrococcus Furiosus ◽  
Single Type ◽  
Kinetic Properties ◽  
Significant Similarity ◽  
Reading Frame ◽  
Hyperthermophilic Archaeon ◽  
Terminal Amino

ABSTRACT Glucose-6-phosphate isomerase (phosphoglucose isomerase [PGI]) (EC 5.3.1.9 ) from the hyperthermophilic archaeon Pyrococcus furiosus was purified 500-fold to homogeneity. The enzyme had an apparent molecular mass of 43 kDa and was composed of a single type of subunit of 23 kDa indicating a homodimeric (α2) structure. Kinetic constants of the enzyme were determined at the optimal pH 7 and at 80°C. Rate dependence on both substrates followed Michaelis-Menten kinetics. The apparent Km values for glucose-6-phosphate and fructose-6-phosphate were 8.7 and 1.0 mM, respectively, and the corresponding apparentV max values were 800 and 130 U/mg. The enzyme had a temperature optimum of 96°C and showed a significant thermostability up to 100°C, which is in accordance with its physiological function under hyperthermophilic conditions. Based on the N-terminal amino acid sequence of the subunit, a single open reading frame (ORF; Pf_209264) was identified in the genome of P. furiosus. The ORF was characterized by functional overexpression in Escherichia coli as a gene, pgi, encoding glucose-6-phosphate isomerase. The recombinant PGI was purified and showed molecular and kinetic properties almost identical to those of the native PGI purified from P. furiosus. The deduced amino acid sequence of P. furiosus PGI did not reveal significant similarity to the conserved PGI superfamily of eubacteria and eucarya. This is the first description of an archaeal PGI, which represents a novel type of PGI.

scholarly journals Characterization of Native and Recombinant Forms of an Unusual Cobalt-Dependent Proline Dipeptidase (Prolidase) from the Hyperthermophilic Archaeon Pyrococcus furiosus

1998 ◽  
Vol 180 (18) ◽  
pp. 4781-4789 ◽  
Author(s):  
Mousumi Ghosh ◽  
Amy M. Grunden ◽  
Dianne M. Dunn ◽  
Robert Weiss ◽  
Michael W. W. Adams
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Substrate Specificity ◽  
Metal Ion ◽  
Pyrococcus Furiosus ◽  
Cobalt Atom ◽  
Significant Similarity ◽  
Genome Database ◽  
Hyperthermophilic Archaeon ◽  
Cell Extracts

ABSTRACT Proline dipeptidase (prolidase) was purified from cell extracts of the proteolytic, hyperthermophilic archaeon Pyrococcus furiosus by multistep chromatography. The enzyme is a homodimer (39.4 kDa per subunit) and as purified contains one cobalt atom per subunit. Its catalytic activity also required the addition of Co2+ ions (Kd , 0.24 mM), indicating that the enzyme has a second metal ion binding site. Co2+could be replaced by Mn2+ (resulting in a 25% decrease in activity) but not by Mg2+, Ca2+, Fe2+, Zn2+, Cu2+, or Ni2+. The prolidase exhibited a narrow substrate specificity and hydrolyzed only dipeptides with proline at the C terminus and a nonpolar amino acid (Met, Leu, Val, Phe, or Ala) at the N terminus. Optimal prolidase activity with Met-Pro as the substrate occurred at a pH of 7.0 and a temperature of 100°C. The N-terminal amino acid sequence of the purified prolidase was used to identify in the P. furiosus genome database a putative prolidase-encoding gene with a product corresponding to 349 amino acids. This gene was expressed in Escherichia coli and the recombinant protein was purified. Its properties, including molecular mass, metal ion dependence, pH and temperature optima, substrate specificity, and thermostability, were indistinguishable from those of the native prolidase from P. furiosus. Furthermore, theKm values for the substrate Met-Pro were comparable for the native and recombinant forms, although the recombinant enzyme exhibited a twofold greaterV max value than the native protein. The amino acid sequence of P. furiosus prolidase has significant similarity with those of prolidases from mesophilic organisms, but the enzyme differs from them in its substrate specificity, thermostability, metal dependency, and response to inhibitors. The P. furiosus enzyme appears to be the second Co-containing member (after methionine aminopeptidase) of the binuclear N-terminal exopeptidase family.

scholarly journals Molecular Identification of Family 38 α-Mannosidase of Bacillus sp. Strain GL1, Responsible for Complete Depolymerization of Xanthan

2002 ◽  
Vol 68 (6) ◽  
pp. 2731-2736 ◽  
Author(s):  
Hirokazu Nankai ◽  
Wataru Hashimoto ◽  
Kousaku Murata
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Cell Extract ◽  
Amino Acid Sequences ◽  
Glycoside Hydrolase Family ◽  
Sequence Information ◽  
Reading Frame ◽  
A Cell ◽  
Terminal Amino ◽  
Amino Acid Sequence Information

ABSTRACT When cells of Bacillus sp. strain GL1 were grown in a medium containing xanthan as a carbon source, α-mannosidase exhibiting activity toward p-nitrophenyl-α-d-mannopyranoside (pNP-α-d-Man) was produced intracellularly. The 350-kDa α-mannosidase purified from a cell extract of the bacterium was a trimer comprising three identical subunits, each with a molecular mass of 110 kDa. The enzyme hydrolyzed pNP-α-d-Man (Km = 0.49 mM) and d-mannosyl-(α-1,3)-d-glucose most efficiently at pH 7.5 to 9.0, indicating that the enzyme catalyzes the last step of the xanthan depolymerization pathway of Bacillus sp. strain GL1. The gene for α-mannosidase cloned most by using N-terminal amino acid sequence information contained an open reading frame (3,144 bp) capable of coding for a polypeptide with a molecular weight of 119,239. The deduced amino acid sequence showed homology with the amino acid sequences of α-mannosidases belonging to glycoside hydrolase family 38.

scholarly journals High sequence homology between protein tyrosine acid phosphatase from boar seminal vesicles and human prostatic acid phosphatase.

2009 ◽  
Vol 56 (3) ◽  
Author(s):  
Paweł Wysocki ◽  
Grazyna Płucienniczak ◽  
Jerzy Strzezek
Keyword(s):  
Acid Phosphatase ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Seminal Vesicle ◽  
Prostatic Acid Phosphatase ◽  
Kinetic Properties ◽  
Terminal Amino Acid ◽  
Protein Tyrosine ◽  
Terminal Amino ◽  
Terminal Amino Acid Sequence

Boar seminal vesicle protein tyrosine acid phosphatase (PTAP) and human prostatic acid phosphatase (PAP) show high affinity for protein phosphotyrosine residues. The physico-chemical and kinetic properties of the boar and human enzymes are different. The main objective of this study was to establish the nucleotide sequence of cDNA encoding boar PTAP and compare it with that of human PAP cDNA. Also, the amino-acid sequence of boar PTAP was compared with the sequence of human PAP. PTAP was isolated from boar seminal vesicle fluid and sequenced. cDNA to boar seminal vesicle RNA was synthesized, amplified by PCR, cloned in E. coli and sequenced. The obtained N-terminal amino-acid sequence of boar PTAP showed 92% identity with the N-terminal amino-acid sequence of human PAP. The determined sequence of a 354 bp nucleotide fragment (GenBank accession number: GQ184596) showed 90% identity with the corresponding sequence of human PAP. On the basis of this sequence a 118 amino acid fragment of boar PTAP was predicted. This fragment showed 89% identity with the corresponding fragment of human PAP and had a similar hydropathy profile. The compared sequences differ in terms of their isoelectric points and amino-acid composition. This may explain the differences in substrate specificity and inhibitor resistance of boar PTAP and human PAP.

The glutamate dehydrogenase-encoding gene of the hyperthermophilic archaeon Pyrococcus furiosus: sequence, transcription and analysis of the deduced amino acid sequence

Gene ◽  
1993 ◽  
Vol 132 (1) ◽  
pp. 143-148 ◽  
Author(s):  
Rik I.L. Eggen ◽  
Ans C.M. Geerling ◽  
Kerstin Waldkötter ◽  
Garabed Antranikian ◽  
Willem M. de Vos
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Glutamate Dehydrogenase ◽  
Pyrococcus Furiosus ◽  
Hyperthermophilic Archaeon ◽  
Encoding Gene

scholarly journals 2,4′-Dihydroxyacetophenone dioxygenase (EC 1.13.11.41) from Alcaligenes sp. 4HAP: a novel enzyme with an atypical dioxygenase sequence

1999 ◽  
Vol 344 (2) ◽  
pp. 397-402 ◽  
Author(s):  
David J. HOPPER ◽  
Mustak A. KADERBHAI
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Predicted Amino Acid Sequence ◽  
Sequencing Data ◽  
Amino Acid Sequencing ◽  
Terminal Amino Acid ◽  
Significant Similarity ◽  
Sds Page ◽  
C And N ◽  
Terminal Amino

2,4′-Dihydroxyacetophenone dioxygenase (EC 1.13.11.41) was purified to homogeneity from Alcaligenes sp. 4HAP grown on 4-hydroxyacetophenone. Measurements of the Mr of the native enzyme ranged from 81600 to 87000, whereas values of 21000 and 20379 were given by SDS/PAGE and electrospray MS respectively. The enzyme is a homotetramer and contains one atom of iron per molecule of enzyme. From C- and N-terminal analyses, primers for PCR were designed and the dad gene cloned and sequenced. The predicted amino acid sequence of dad, deduced from the nucleotide sequence, confirms the N-terminal amino acid sequencing data and contains the sequence of an internal tryptic peptide. It gave a calculated Mr of 20364. The gene was expressed in Escherichia coli and yielded active enzyme. The derived amino acid sequence does not show significant similarity to other dioxygenases or any strong similarity to protein sequences presently available in the databases.

C-Terminal amino acid sequence of chicken pepsinogen and its homology with sequences of other acid proteases

1980 ◽  
Vol 45 (4) ◽  
pp. 1144-1154 ◽  
Author(s):  
Miroslav Baudyš ◽  
Helena Keilová ◽  
Vladimír Kostka
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
The Other ◽  
Terminal Sequence ◽  
Terminal Part ◽  
Terminal Amino Acid ◽  
Tryptic Peptides ◽  
Terminal Amino ◽  
High Degree ◽  
Terminal Amino Acid Sequence

To determine the primary structure of the C-terminal part of the molecule of chicken pepsinogen the tryptic, chymotryptic and thermolytic digest of the protein were investigated and peptides derived from this region were sought. These peptides permitted the following 21-residue C-terminal sequence to be determined: ...Ile-Arg-Glu-Tyr-Tyr-Val-Ile-Phe-Asp-Arg-Ala-Asn-Asn-Lys-Val-Gly-Leu-Ser-Pro-Leu-Ser.COOH. A comparison of this structure with the C-terminal sequential regions of the other acid proteases shows a high degree of homology between chicken pepsinogen and these proteases (e.g., the degree of homology with respect to hog pepsinogen and calf prochymosin is about 66%). Additional tryptic peptides, derived from the N-terminal part of the zymogen molecule whose amino acid sequence has been reported before, were also obtained in this study. This sequence was extended by two residues using an overlapping peptide. An ancillary result of this study was the isolation of tryptic peptides derived from other regions of the zymogen molecule.

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