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1.
Proc Natl Acad Sci U S A ; 98(14): 7835-40, 2001 Jul 03.
Article in English | MEDLINE | ID: mdl-11427726

ABSTRACT

The genome of the crenarchaeon Sulfolobus solfataricus P2 contains 2,992,245 bp on a single chromosome and encodes 2,977 proteins and many RNAs. One-third of the encoded proteins have no detectable homologs in other sequenced genomes. Moreover, 40% appear to be archaeal-specific, and only 12% and 2.3% are shared exclusively with bacteria and eukarya, respectively. The genome shows a high level of plasticity with 200 diverse insertion sequence elements, many putative nonautonomous mobile elements, and evidence of integrase-mediated insertion events. There are also long clusters of regularly spaced tandem repeats. Different transfer systems are used for the uptake of inorganic and organic solutes, and a wealth of intracellular and extracellular proteases, sugar, and sulfur metabolizing enzymes are encoded, as well as enzymes of the central metabolic pathways and motility proteins. The major metabolic electron carrier is not NADH as in bacteria and eukarya but probably ferredoxin. The essential components required for DNA replication, DNA repair and recombination, the cell cycle, transcriptional initiation and translation, but not DNA folding, show a strong eukaryal character with many archaeal-specific features. The results illustrate major differences between crenarchaea and euryarchaea, especially for their DNA replication mechanism and cell cycle processes and their translational apparatus.


Subject(s)
Genome, Archaeal , Sulfolobus/genetics , Cell Cycle Proteins/genetics , DNA Replication , Molecular Sequence Data , Sequence Analysis, DNA
2.
Extremophiles ; 4(3): 175-9, 2000 Jun.
Article in English | MEDLINE | ID: mdl-10879562

ABSTRACT

The translational starts of 144 Sulfolobus solfataricus genes have been determined by database comparison. Half the genes lie inside operons and the other half are at the start of an operon or single genes. A Shine-Dalgarno sequence is found upstream of the genes inside operons, but not for the first gene in an operon or isolated genes; this indicates that two different mechanisms are used for translation initiation in S. solfataricus. A box A transcriptional signal is found for the genes starting an operon or isolated genes, but not for the genes inside an operon. The box A signal is located about 27 nt upstream of the start codon, which implies that little or no upstream sequence is available for translation initiation for this group of genes. This finding is discussed.


Subject(s)
Peptide Chain Initiation, Translational , Sulfolobus/genetics , Base Sequence , Codon, Initiator/genetics , DNA, Archaeal/genetics , Genes, Archaeal , Molecular Sequence Data , Operon , Promoter Regions, Genetic , RNA, Archaeal/genetics , RNA, Ribosomal, 16S/genetics
3.
Extremophiles ; 2(3): 305-12, 1998 Aug.
Article in English | MEDLINE | ID: mdl-9783178

ABSTRACT

The Sulfolobus solfataricus P2 genome collaborators are poised to sequence the entire 3-Mbp genome of this crenarchaeote archaeon. About 80% of the genome has been sequenced to date, with the rest of the sequence being assembled fast. In this publication we introduce the genomic sequencing and automated analysis strategy and present intial data derived from the sequence analysis. After an overview of the general sequence features, metabolic pathway studies are explained, using sugar metabolism as an example. The paper closes with an overview of repetitive elements in S. solfataricus.


Subject(s)
Genome , Sulfolobus/genetics , Base Sequence , Carbohydrate Metabolism , Chromosome Mapping , Cloning, Molecular , DNA, Archaeal/genetics , Genes, Archaeal , Phylogeny , Repetitive Sequences, Nucleic Acid , Sequence Analysis, DNA , Software , Sulfolobus/classification , Sulfolobus/metabolism
4.
Protein Eng ; 9(6): 519-24, 1996 Jun.
Article in English | MEDLINE | ID: mdl-8862552

ABSTRACT

The importance of Glu87 and Trp89 in the lid of Humicola lanuginosa lipase for the hydrolytic activity at the water/lipid interface was investigated by site-directed mutagenesis. It was found that the effect on the hydrolytic activity upon the replacement of Trp89 with Phe, Leu, Gly or Glu was substrate dependent. The Trp89 mutants displayed an altered chain length specificity towards triglycerides, with a higher relative activity towards triacetin and trioctanoin compared with tributyrin. Trp89 was shown to be less important in the hydrolysis of vinyl esters compared with ethyl esters and triglycerides. An exclusive effect on the acylation reaction rate by the mutation of Trp89 was consistent with the data. It is suggested that Trp89 is important in the process of binding the acyl chain of the substrate into the active site for optimal acylation reaction rate. The Trp89Phe mutation resulted in an increased hydrolytic activity towards 2-alkylalkanoic acid esters. This is suggested to be due to reduction of unfavourable van der Waals contacts between Trp89 and the 2-substituent of the substrate. Thus, in contrast to natural substrates, Trp89 has a negative impact on the catalytic efficiency when substrates with bulky acyl chains are used. In contrast to the Trp89 mutations, the effect on the hydrolytic activity of the Glu87Ala mutation was almost substrate independent, 35-70% activity of wild-type lipase. A reduction of both the acylation and deacylation reaction was consistent with the data.


Subject(s)
Fungal Proteins/chemistry , Glutamic Acid/chemistry , Lipase/chemistry , Mitosporic Fungi/enzymology , Models, Molecular , Protein Conformation , Tryptophan/chemistry , Acylation , Binding Sites , Chemical Phenomena , Chemistry, Physical , Esters/metabolism , Fungal Proteins/genetics , Fungal Proteins/metabolism , Hydrolysis , Lipase/genetics , Lipase/metabolism , Mitosporic Fungi/genetics , Mutagenesis, Site-Directed , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Substrate Specificity , Triglycerides/chemistry , Triglycerides/metabolism
5.
Mol Gen Genet ; 247(4): 423-9, 1995 May 20.
Article in English | MEDLINE | ID: mdl-7770049

ABSTRACT

A region of the Aspergillus nidulans genome carrying the sA and sC genes, encoding PAPS reductase and ATP sulphurylase, respectively, was isolated by transformation of an sA mutant with a cosmid library. The genes were subcloned and their functions confirmed by retransformation and complementation of A. nidulans strains carrying sA and sC mutations. The physical distance of 2 kb between the genes corresponds to a genetic distance of 1 cM. While the deduced amino acid sequence of the sA gene product shows homology with the equivalent MET16 gene product of Saccharomyces cerevisiae, the sC gene product resembles the equivalent MET3 yeast gene product at the N-terminal end, but differs markedly from it at the C-terminal end, showing homology to the APS kinases of several microorganisms. It is proposed that this C-terminal region does not encode a functional APS kinase, but is responsible for allosteric regulation by PAPS of the sulphate assimilation pathway in A. nidulans, and that the ATP sulphurylase encoding-gene (sC) of filamentous ascomycetes may have evolved from a bifunctional gene similar to the nodQ gene of Rhizobium meliloti.


Subject(s)
Aspergillus nidulans/genetics , Genes, Fungal , Sulfate Adenylyltransferase/genetics , Sulfates/metabolism , Adenosine Triphosphate/metabolism , Allosteric Regulation , Amino Acid Sequence , Cloning, Molecular , Cosmids , DNA, Fungal/genetics , Molecular Sequence Data , Oxidation-Reduction , Sequence Alignment , Sequence Homology, Amino Acid
6.
J Protein Chem ; 14(4): 217-24, 1995 May.
Article in English | MEDLINE | ID: mdl-7662109

ABSTRACT

To reveal the functional role of Glu87 and Trp89 in the lid of Humicola lanuginosa lipase, site-directed mutagenesis at Glu87 and Trp89 was carried out. The catalytic performance of wild-type and mutated lipases was studied in transesterification reactions in cyclohexane at a controlled water activity. Two different acyl donors were used in the investigation: tributyrin, a natural substrate for a lipase, and vinyl butyrate, an activated ester suitable for fast and efficient lipase-catalyzed transformations in preparative organic synthesis. As acyl acceptor 1-heptanol was used. The Glu87Ala mutation decreased the Vmax,app value with tributyrin and vinyl butyrate by a factor of 1.5 and 2, respectively. The Km,app for tributyrin was not affected by the Glu87Ala mutation, but the Km,app for vinyl butyrate increased twofold compared to the wild-type lipase. Changing Trp89 into a Phe residue afforded an enzyme with a 2.7- and 2-fold decreased Vmax,app with the substrates tributyrin and vinyl butyrate, respectively, compared to the wild-type lipase. No significant effects on the Km,app values for tributyrin or vinyl butyrate were seen as a result of the Trp89Phe mutation. However, the introduction of a Glu residue at position 89 in the lid increased the Km,app for tributyrin and vinyl butyrate by a factor of > 5 and 2, respectively. The Trp89Glu mutated lipase could not be saturated with tributyrin within the experimental conditions (0-680 mM) studied here. With vinyl butyrate as a substrate the Vmax,app was only 6% of that obtained with wild-type enzyme.


Subject(s)
Glutamine , Lipase/chemistry , Mitosporic Fungi/enzymology , Tryptophan , Binding Sites , Butyrates/metabolism , Cyclohexanes , Esterification , Kinetics , Lipase/genetics , Lipase/metabolism , Models, Molecular , Mutagenesis, Site-Directed , Solvents , Structure-Activity Relationship , Substrate Specificity , Triglycerides/metabolism , Vinyl Compounds/metabolism
8.
Lipids ; 29(9): 599-603, 1994 Sep.
Article in English | MEDLINE | ID: mdl-7815893

ABSTRACT

To determine whether Trp89 located in the lid of the lipase (EC 3.1.1.3) from Humicola lanuginosa is important for the catalytic property of the enzyme, site-directed mutagenesis at Trp89 was carried out. The kinetic properties of wild type and mutated enzymes were studied with tributyrin as substrate. Lipase variants in which Trp89 was changed to Phe, Leu, Gly or Glu all showed less than 14% of the activity compared to that of the wild type lipase. The Trp89Glu mutant was the least active with only 1% of the activity seen with the wild type enzyme. All Trp mutants had the same binding affinity to the tributyrin substrate interface as did the wild type enzyme. Wild type lipase showed saturation kinetics against tributyrin when activities were measured with mixed emulsions containing different proportions of tributyrin and the nonionic alkyl polyoxyethylene ether surfactant, Triton DF-16. Wild type enzyme showed a Vmax = 6000 +/- 300 mmol.min-1.g-1 and an apparent Km = 16 +/- 2% (vol/vol) for tributyrin in Triton DF-16, while the mutants did not show saturation kinetics in an identical assay. The apparent Km for tributyrin in Triton DF-16 was increased as the result of replacing Trp89 with other residues (Phe, Leu, Gly or Glu). The activities of all mutants were more sensitive to the presence of Triton DF-16 in the tributyrin substrate than was wild type lipase. The activity of the Trp89Glu mutant was decreased to 50% in the presence of 2 vol% Triton DF-16 compared to the activity seen with pure tributyrin as substrate.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Lipase/metabolism , Mitosporic Fungi/enzymology , Triglycerides/metabolism , Detergents/metabolism , Emulsions , Hydrolysis , Kinetics , Lipase/chemistry , Lipase/genetics , Mitosporic Fungi/genetics , Models, Biological , Mutagenesis, Site-Directed , Organic Chemicals , Tryptophan/chemistry , Tryptophan/genetics
9.
J Protein Chem ; 12(6): 749-57, 1993 Dec.
Article in English | MEDLINE | ID: mdl-8136025

ABSTRACT

The homologous lipases from Rhizomucor miehei and Humicola lanuginosa showed approximately the same enantioselectivity when 2-methyldecanoic acid esters were used as substrates. Both lipases preferentially hydrolyzed the S-enantiomer of 1-heptyl 2-methyldecanoate (R. miehei: ES = 8.5; H. lanuginosa: ES = 10.5), but the R-enantiomer of phenyl 2-methyldecanoate (ER = 2.9). Chemical arginine specific modification of the R. miehei lipase with 1,2-cyclohexanedione resulted in a decreased enantioselectivity (ER = 2.0), only when the phenyl ester was used as a substrate. In contrast, treatment with phenylglyoxal showed a decreased enantioselectivity (ES = 2.5) only when the heptyl ester was used as a substrate. The presence of guanidine, an arginine side chain analog, decreased the enantioselectivity with the heptyl ester (ES = 1.9) and increased the enantioselectivity with the aromatic ester (ER = 4.4) as substrates. The mutation, Glu 87 Ala, in the lid of the H. lanuginosa lipase, which might decrease the electrostatic stabilization of the open-lid conformation of the lipase, resulted in 47% activity compared to the native lipase, in a tributyrin assay. The Glu 87 Ala mutant showed an increased enantioselectivity with the heptyl ester (ES = 17.4) and a decreased enantioselectivity with the phenyl ester (ER = 2.5) as substrates, compared to native lipase. The enantioselectivities of both lipases in the esterification of 2-methyldecanoic acid with 1-heptanol were unaffected by the lid modifications.


Subject(s)
Enzymes, Immobilized/metabolism , Lipase/metabolism , Mitosporic Fungi/enzymology , Mucorales/enzymology , Arginine , Cyclohexanones/pharmacology , Decanoates/metabolism , Hydrolysis , Kinetics , Phenylglyoxal/pharmacology , Stereoisomerism , Substrate Specificity
10.
Protein Eng ; 2(6): 449-57, 1989 Mar.
Article in English | MEDLINE | ID: mdl-2652140

ABSTRACT

Californium-252 plasma desorption mass spectrometry (PDMS) has been employed for the characterization of a series of human insulin derivatives in order to evaluate the performance of this technique as an analytical tool in protein engineering. Several of the characterized modifications result in a 1 a.m.u. mass change. The precision in mass determination obtainable by PDMS analysis is not sufficient for unambiguous verification of such modifications based on the molecular weight alone. It is, however, possible to carry out in situ enzymatic digestion of the sample. Subsequent PDMS analysis will in most cases reveal if the modification has been introduced as intended.


Subject(s)
Insulin/analysis , Protein Engineering , Amino Acid Sequence , Humans , Mass Spectrometry , Molecular Sequence Data , Molecular Weight
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