"Short-chain" alpha-1,4-glucan phosphorylase having a truncated N-terminal domain: functional expression and characterization of the enzyme from Sulfolobus solfataricus.
All known alpha-1,4-glucan phosphorylases (GPs) are active as homodimers and use their N-terminal domains for oligomerisation. Structure-based sequence comparison of a putative phosphorylase from the thermophilic crenarchaeon Sulfolobus solfataricus (SsGP) with the well characterized GP from Escherichia coli reveals that SsGP totally lacks the otherwise conserved regions for building the dimer interface. Because all efforts of producing functional SsGP in E. coli failed, we used heterologous gene expression in the hyperthermophilic archaeon Thermococcus kodakaraensis and isolated, in low amounts, SsGP harboring Strep-Tag II fused to the C-terminal Tyr-465 of the enzyme. The recombinant protein eluted in size exclusion chromatography with an apparent molecular mass of approximately 69 kDa, consistent with neither the mass expected for a monomer (55 kDa) nor that of a homodimer (110 kDa). The biochemical properties of SsGP were similar to those seen for other GPs containing the N-terminal elements for dimerisation, suggesting that the "short-chain" format of SsGP is fully appropriate for phosphorylase catalytic function and stability. However, the substrate specificity of SsGP differed from that reported for GPs from other thermophilic microorganisms.
Mueller
Mario
M
Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12, A-8010 Graz, Austria.
Takemasa
Ryo
R
Schwarz
Alexandra
A
Atomi
Haruyuki
H
Nidetzky
Bernd
B
D000595:Amino Acid Sequence; D004926:Escherichia coli; D019848:Gene Expression Regulation, Archaeal; D015971:Gene Expression Regulation, Enzymologic; D005936:Glucans; D005964:Glucosyltransferases / Q000235:genetics; D007700:Kinetics; D006005:Phosphorylases / Q000096:biosynthesis* / Q000235:genetics*; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D048229:Sulfolobus solfataricus / Q000201:enzymology*; D019712:Thermococcus / Q000201:enzymology
D005936:Glucans; D011994:Recombinant Proteins; C040088:1,4-glucan; D005964:Glucosyltransferases; D006005:Phosphorylases; C033938:maltodextrin phosphorylase