Characterisation of a highly stable alpha-amylase from the halophilic archaeon Haloarcula hispanica.
Intracellular and extracellular proteins from halophilic archaea face very saline conditions and must be able to maintain stability and functionality at nearly saturated salt concentrations. Haloarchaeal proteins contain specific adaptations to prevent aggregation and loss of activity in such conditions, but these adaptations usually result in a lack of stability in the absence of salt. Here, we present the characterisation of a secreted alpha-amylase (AmyH) from the halophilic archaeon Haloarcula hispanica. AmyH was shown to be very halophilic but, unusually for a halophilic protein, it retained activity in the absence of salt. Intrinsic fluorescence measurements and activity assays showed that AmyH was very stable in high-salt buffer and even maintained stability upon the addition of urea. Urea-induced denaturation was only achieved in the absence of NaCl, demonstrating clearly that the stability of the protein was salt-dependent. Sequencing of the amyH gene showed an amino acid composition typical of halophilic proteins and, moreover, the presence of a signal peptide containing diagnostic features characteristic of export via the Twin-arginine translocase (Tat). Analysis of the export of AmyH showed that it was translocated post-translationally, most likely in a folded and active conformation, confirming that AmyH is a substrate of the Tat pathway.
Hutcheon
George W
GW
Department of Biological Sciences, University of Warwick, CV4 7AL, Coventry, UK.
Vasisht
Nishi
N
Bolhuis
Albert
A
D000595:Amino Acid Sequence; D000681:Amylases / Q000737:chemistry; D019843:Archaeal Proteins / Q000737:chemistry* / Q000378:metabolism; D015153:Blotting, Western; D002122:Calcium Chloride / Q000494:pharmacology; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D029968:Escherichia coli Proteins / Q000737:chemistry; D019613:Haloarcula / Q000201:enzymology*; D008027:Light; D026901:Membrane Transport Proteins / Q000737:chemistry; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017510:Protein Folding; D021382:Protein Sorting Signals; D021381:Protein Transport; D012542:Scattering, Radiation; D017386:Sequence Homology, Amino Acid; D012965:Sodium Chloride / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013696:Temperature; D014508:Urea / Q000494:pharmacology; D000516:alpha-Amylases / Q000737:chemistry*
D019843:Archaeal Proteins; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D021382:Protein Sorting Signals; C408585:twin-arginine translocase complex, E coli; D012965:Sodium Chloride; D014508:Urea; D000681:Amylases; D000516:alpha-Amylases; D002122:Calcium Chloride