ABSTRACT
A cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) was successfully isolated and characterized from the halophilic archaeon Haloferax mediterranei. The enzyme is a monomer with a molecular mass of 77 kDa and optimum activity at 55°C, pH 7.5 and 1.5 M NaCl. The enzyme displayed many activities related to the degradation and transformation of starch. Cyclization was found to be the predominant activity, yielding a mixture of cyclodextrins, mainly α-CD, followed by hydrolysis and to a lesser extent coupling and disproportionation activities. Gene encoding H. mediterranei CGTase was cloned and heterologously overexpressed. Sequence analysis revealed an open reading frame of 2142 bp that encodes a protein of 713 amino acids. The amino acid sequence displayed high homology with those belonging to the α-amylase family. The CGTase is secreted to the extracellular medium by the Tat pathway. Upstream of the CGTase gene, four maltose ABC transporter genes have been sequenced (malE, malF, malG, malK). The expression of the CGTase gene yielded a fully active CGTase with similar kinetic behavior to the wild-type enzyme. The H. mediterranei CGTase is the first halophilic archaeal CGTase characterized, sequenced and expressed.
Subject(s)
Glucosyltransferases/metabolism , Haloferax mediterranei/enzymology , Starch/metabolism , Chromatography, Liquid , Cyclization , Electrophoresis, Polyacrylamide Gel , Glucosyltransferases/chemistry , Haloferax mediterranei/metabolism , Hydrogen-Ion Concentration , Hydrolysis , Kinetics , Molecular Weight , Spectrometry, Mass, Electrospray Ionization , Substrate Specificity , TemperatureABSTRACT
NifS-like proteins are pyridoxal 5'-phosphate (PLP)-dependent enzymes involved in sulphur transfer metabolism. These enzymes have been catalogued as cysteine desulphurases (CDs) which catalyse the conversion of L-cysteine into L-alanine and an enzyme-bound persulphide radical. This reaction, assisted by different scaffold protein machineries, seems to be the main source of sulphur for the synthesis of essential cofactors of the [Fe-S] cluster. CDs genes have been detected in the tree domains of life, but, up until now, there has been no biochemical characterisation or study into the physiological role of this enzyme in haloarchaea. In this study, we have cloned, expressed and characterised a cysteine desulphurase (SufS) from Haloferax volcanii and demonstrated that this protein is able to reconstitute the [Fe-S] cluster of halophilic ferredoxin.
Subject(s)
Archaea/metabolism , Gene Expression Regulation, Bacterial , Haloferax/metabolism , Iron-Sulfur Proteins/chemistry , Lyases/genetics , Lyases/metabolism , DNA Primers/chemistry , Databases, Protein , Dose-Response Relationship, Drug , Electrophoresis, Polyacrylamide Gel , Escherichia coli/metabolism , Hydrogen-Ion Concentration , Nucleotides/chemistry , Protein Folding , Recombinant Proteins/chemistry , Ultraviolet RaysABSTRACT
The NAD-dependent glutamate dehydrogenase (GDH) gene from the halophilic archaeon Haloferax mediterranei has been cloned. The analysis of the nucleotide sequence revealed an open reading frame of 1323 bp that encodes a NAD-GDH. The amino acid sequence displayed high homology with those from other sources, especially the highly conserved residues involved in 2-oxoglutarate binding. The expression of this gene in Escherichia coli, the refolding and further characterization, yielded a fully active NAD-GDH with the same features than those found for the wild-type enzyme. This halophilic NAD-GDH showed a highly dependence on salts for both stability and activity, being essential for the refolding of the recombinant enzyme.
Subject(s)
Glutamate Dehydrogenase/chemistry , Haloferax mediterranei/enzymology , Haloferax mediterranei/genetics , Amino Acid Sequence , Archaeal Proteins/chemistry , Archaeal Proteins/genetics , Archaeal Proteins/metabolism , Cloning, Molecular , Gene Library , Glutamate Dehydrogenase/genetics , Glutamate Dehydrogenase/metabolism , Hydrogen-Ion Concentration , Molecular Sequence Data , Polymerase Chain Reaction , Protein Folding , Recombinant Proteins , Sequence Homology, Amino Acid , Substrate Specificity , TemperatureABSTRACT
Salinibacter ruber, an extremely halophilic member of the domain Bacteria, has two different cytoplasmic glutamate dehydrogenase activities, marked as GDHI and GDHII. GDHI showed a strong dependence on high salt concentrations for stability, but not for activity, displaying maximal activity in the absence of salts. GDHII depended on high salt concentrations for both activity and stability. It catalyzed amination of 2-oxoglutarate with optimal activity in 3 M KCl at pH 8. No activating effect was found when NaCl was replaced by KCl. Only GDHII displayed activity in the deamination reaction of glutamate with an optimal pH of 9.5. Both enzymes were activated by certain amino acids (L-leucine, L-histidine, L-phenylalanine) and by nucleotides such as ADP or ATP. A low-molecular-mass cytoplasmic fraction was found to be a highly effective activator of GDHII in the presence of high NaCl concentrations.
Subject(s)
Bacteroidetes/enzymology , Glutamate Dehydrogenase/isolation & purification , Glutamate Dehydrogenase/metabolism , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , Bacterial Proteins/isolation & purification , Bacterial Proteins/metabolism , Enzyme Activation , Enzyme Stability/drug effects , Glutamic Acid/metabolism , Histidine/metabolism , Hydrogen-Ion Concentration , Ketoglutaric Acids/metabolism , Leucine/metabolism , NAD/metabolism , NADP/metabolism , Phenylalanine/metabolism , Potassium Chloride , Sodium Chloride , Substrate SpecificityABSTRACT
Sulfolobus solfataricus secretes an acid-resistant alpha-amylase (amyA) during growth on starch as the sole carbon and energy source. Synthesis of this activity is subject to catabolite repression. To better understand alpha-amylase function and regulation, the structural gene was identified and disrupted and the resulting mutant was characterized. Internal alpha-amylase peptide sequences obtained by tandem mass spectroscopy were used to identify the amyA coding sequence. Anti-alpha-amylase antibodies raised against the purified protein immunoprecipitated secreted alpha-amylase activity and verified the enzymatic identity of the sequenced protein. A new gene replacement method was used to disrupt the amyA coding sequence by insertion of a modified allele of the S. solfataricus lacS gene. PCR and DNA sequence analysis were used to characterize the altered amyA locus in the recombinant strain. The amyA::lacS mutant lost the ability to grow on starch, glycogen, or pullulan as sole carbon and energy sources. During growth on a non-catabolite-repressing carbon source with added starch, the mutant produced no detectable secreted amylase activity as determined by enzyme assay, plate assay, or Western blot analysis. These results clarify the biological role of the alpha-amylase and provide additional methods for the directed genetic manipulation of the S. solfataricus genome.
Subject(s)
Gene Targeting , Hot Temperature , Mutation , Sulfolobus/enzymology , alpha-Amylases/genetics , alpha-Amylases/metabolism , Amino Acid Sequence , Culture Media , Gene Expression Regulation, Archaeal , Molecular Sequence Data , Recombination, Genetic , Spectrometry, Mass, Electrospray Ionization , Sulfolobus/genetics , Sulfolobus/growth & development , alpha-Amylases/chemistryABSTRACT
An electric water heater was modified for large-scale cultivation of aerobic acidophilic hyperthermophiles to enable recovery of secreted proteins. Critical changes included thermostat replacement, redesign of the temperature control circuit, and removal of the cathodic anticorrosion system. These alterations provided accurate temperature and pH control. The bioreactor was used to cultivate selected strains of the archaeon Sulfolobus solfataricus and other species within this genus. Reformulation of a basal salts medium facilitated preparation of large culture volumes and eliminated sterilization-induced precipitation of medium components. Substrate induction of synthesis of the S. solfataricus-secreted alpha-amylase during growth in a defined medium supported the utility of the bioreactor for studies of physiologically regulated processes. An improved purification strategy was developed by using strong cation-exchange chromatography for recovery of the alpha-amylase and the processing of large sample volumes of acidic culture supernatant. These findings should simplify efforts to study acidophilic hyperthermophilic microbes and their secreted proteins.
