Abstract
We report the heterologous expression and molecular characterization of the first extremely halophilic alpha-glucosidase (EC 3.2.1.20) from the archaeon Haloquadratum walsbyi. A 2349 bp region (Hqrw_2071) from the Hqr. walsbyi C23 annotated genome was PCR-amplified and the resulting amplicon ligated into plasmid pET28b(+), expressed in E. coli Rosetta cells, and the resulting protein purified by Ni-NTA affinity chromatography. The recombinant protein showed an estimated molecular mass of 87 kDa, consistent with the expected value of the annotated protein, and an optimal activity for the hydrolysis of α-PNPG was detected at 40 °C, and at pH 6.0. Enzyme activity values were the highest in the presence of 3 M NaCl or 3–4 M KCl. However, specific activity values were two-fold higher in the presence of 3–4 M KCl when compared to NaCl suggesting a cytoplasmic localization. Phylogenetic analyses, with respect to other alpha-glucosidases from members of the class Halobacteria, showed that the Hqr. walsbyi MalH was most similar (up to 41%) to alpha-glucosidases and alpha-xylosidases of Halorubrum. Moreover, computational analyses for the detection of functional domains, active and catalytic sites, as well as 3D structural predictions revealed a close relationship with an E. coli YicI-like alpha-xylosidase of the GH31 family. However, the purified enzyme did not show alpha-xylosidase activity. This narrower substrate range indicates a discrepancy with annotations from different databases and the possibility of specific substrate adaptations of halophilic glucosidases due to high salinity. To our knowledge, this is the first report on the characterization of an alpha-glucosidase from the halophilic Archaea, which could serve as a new model to gain insights into carbon metabolism in this understudied microbial group.
Highlights
Haloquadratum walsbyi is a squared-shaped, extremely halophilic member of the Euryarchaeota, which has been described as the dominant representative of the microbiota present in aquatic hypersaline (≥32% NaCl) environments [1,2,3]
As many as 43 genes with predicted functions related to carbohydrate metabolism, including a putative alpha-glucosidase encoded by an open reading frame (ORFs) Hqrw_2071 and HQ1911A, have been described in both strains [5]
Protein sequences of alpha-glucosidases from Archaea, Bacteria and Eukarya were retrieved from the Pfam protein family database [25], except that encoded by Hqr. walsbyi C23, which was obtained from the KEGG database [26]
Summary
Haloquadratum walsbyi is a squared-shaped, extremely halophilic member of the Euryarchaeota, which has been described as the dominant representative of the microbiota present in aquatic hypersaline (≥32% NaCl) environments [1,2,3]. Haloquadratum walsbyi as the dominant lineage across a variety of environments with high osmotic stress including hypersaline pools [2,5], salt lakes, and saltern crystallizer ponds. Pure cultures of this archaeon were independently isolated from solar salterns in Spain and Australia by Bolhuis et al [5]. 1,4-alpha-glucosidic linkages of terminal residues of D-glucose in a variety of oligosaccharides [6,7] They are known to carry out the release of glucose from maltose and maltodextrins and to mediate glycoprotein processing in living systems. We describe the cloning and characterization of an alpha-glucosidase gene from Hqr. walsbyi C23, the dominant haloarchaeon in most solar saltern systems according to metagenomic surveys [2]
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