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Molecular Characterization of the α-Glucosidase Gene ( malA ) from the Hyperthermophilic Archaeon Sulfolobus solfataricus

1998; American Society for Microbiology; Volume: 180; Issue: 5 Linguagem: Inglês

10.1128/jb.180.5.1287-1295.1998

1098-5530

Michael Rolfsmeier, Cynthia A. Haseltine, Elisabetta Bini, Amy J. Clark, Paul Blum,

Enzyme Production and Characterization

ABSTRACT Acidic hot springs are colonized by a diversity of hyperthermophilic organisms requiring extremes of temperature and pH for growth. To clarify how carbohydrates are consumed in such locations, the structural gene ( malA ) encoding the major soluble α-glucosidase (maltase) and flanking sequences from Sulfolobus solfataricus were cloned and characterized. This is the first report of an α-glucosidase gene from the archaeal domain. malA is 2,083 bp and encodes a protein of 693 amino acids with a calculated mass of 80.5 kDa. It is flanked on the 5′ side by an unusual 1-kb intergenic region. Northern blot analysis of the malA region identified transcripts for malA and an upstream open reading frame located 5′ to the 1-kb intergenic region. The malA transcription start site was located by primer extension analysis to a guanine residue 8 bp 5′ of the malA start codon. Gel mobility shift analysis of the malA promoter region suggests that sequences 3′ to position −33, including a consensus archaeal TATA box, play an essential role in malA expression. malA homologs were detected by Southern blot analysis in other S. solfataricus strains and in Sulfolobus shibatae , while no homologs were evident in Sulfolobus acidocaldarius , lending further support to the proposed revision of the genus Sulfolobus . Phylogenetic analyses indicate that the closest S. solfataricus α-glucosidase homologs are of mammalian origin. Characterization of the recombinant enzyme purified from Escherichia coli revealed differences from the natural enzyme in thermostability and electrophoretic behavior. Glycogen is a substrate for the recombinant enzyme. Unlike maltose hydrolysis, glycogen hydrolysis is optimal at the intracellular pH of the organism. These results indicate a unique role for the S. solfataricus α-glucosidase in carbohydrate metabolism.