Recombinant production and biochemical characterization of a hyperthermostable alpha-glucan/maltodextrin phosphorylase from Pyrococcus furiosus.
Alpha-glucan phosphorylase catalyzes the reversible cleavage of alpha-1-4-linked glucose polymers into alpha-D-glucose-1-phosphate. We report the recombinant production of an alpha-glucan/maltodextrin phosphorylase (PF1535) from a hyperthermophilic archaeon, Pyrococcus furiosus, and the first detailed biochemical characterization of this enzyme from any archaeal source using a mass-spectrometry-based assay. The apparent 98 kDa recombinant enzyme was active over a broad range of temperatures and pH, with optimal activity at 80 degrees C and pH 6.5-7. This archaeal protein retained its complete activity after 24 h at 80 degrees C in Tris-HCl buffer. Unlike other previously reported phosphorylases, the Ni-affinity column purified enzyme showed broad substrate specificity in both the synthesis and degradation of maltooligosaccharides. In the synthetic direction of the enzymatic reaction, the lowest oligosaccharide required for the chain elongation was maltose. In the degradative direction, the archaeal enzyme can produce glucose-1-phosphate from maltotriose or longer maltooligosaccharides including both glycogen and starch. The specific activity of the enzyme at 80 degrees C in the presence of 10 mM maltoheptaose and at 10 mg ml(-1) glycogen concentration was 52 U mg(-1) and 31 U mg(-1), respectively. The apparent Michaelis constant and maximum velocity for inorganic phosphate were 31 +/- 2 mM and 0.60 +/- 0.02 mM min(-1) microg(-1), respectively. An initial velocity study of the enzymatic reaction indicated a sequential bi-bi catalytic mechanism. Unlike the more widely studied mammalian glycogen phosphorylase, the Pyrococcus enzyme is active in the absence of added AMP.
Mizanur
Rahman M
RM
Department of Chemistry and the Plant Sciences Institute, Gilman Hall, Iowa State University, Ames, IA 50011-3111, USA.
Griffin
Amanda K K
AK
Pohl
Nicola L
NL
D000595:Amino Acid Sequence; D019641:DNA, Archaeal / Q000235:genetics; D004795:Enzyme Stability; D005936:Glucans / Q000378:metabolism*; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D007700:Kinetics; D008969:Molecular Sequence Data; D006005:Phosphorylases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020027:Pyrococcus furiosus / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D021241:Spectrometry, Mass, Electrospray Ionization; D013213:Starch / Q000378:metabolism*; D013379:Substrate Specificity
D019641:DNA, Archaeal; D005936:Glucans; D011994:Recombinant Proteins; D013213:Starch; D005964:Glucosyltransferases; D006005:Phosphorylases; C033938:maltodextrin phosphorylase