A phosphohexomutase from the archaeon Sulfolobus solfataricus is covalently modified by phosphorylation on serine.
A phosphoserine-containing peptide was identified from tryptic digests from Sulfolobus solfataricus P1 by liquid chromatography-tandem mass spectrometry. Its amino acid sequence closely matched that bracketing Ser-309 in the predicted protein product of open reading frame sso0207, a putative phosphohexomutase, in the genome of S. solfataricus P2. Open reading frame sso0207 was cloned, and its protein product expressed in Escherichia coli. The recombinant protein proved capable of interconverting mannose 1-phosphate and mannose 6-phosphate, as well as glucose 1-phosphate and glucose 6-phosphate, in vitro. It displayed no catalytic activity toward glucosamine 6-phosphate or N-acetylglucosamine 6-phosphate. Models constructed using the X-ray crystal structure of a homologous phosphohexomutase from Pseudomonas aeruginosa predicted that Ser-309 of the archaeal protein lies within the substrate binding site. The presence of a phosphoryl group at this location would be expected to electrostatically interfere with the binding of negatively charged phosphohexose substrates, thus attenuating the catalytic efficiency of the enzyme. Using site-directed mutagenesis, Ser-309 was substituted by aspartic acid to mimic the presence of a phosphoryl group. The V(max) of the mutationally altered protein was only 4% that of the unmodified form. Substitution of Ser-309 with larger, but uncharged, amino acids, including threonine, also decreased catalytic efficiency, but to a lesser extent--three- to fivefold. We therefore predict that phosphorylation of the enzyme in vivo serves to regulate its catalytic activity.
Ray
W Keith
WK
Department of Biochemistry and Virginia Institute for Genomics, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061, USA.
Keith
Sabrina M
SM
DeSantis
Andrea M
AM
Hunt
Jeremy P
JP
Larson
Timothy J
TJ
Helm
Richard F
RF
Kennelly
Peter J
PJ
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000378:metabolism*; D001665:Binding Sites; D015971:Gene Expression Regulation, Enzymologic; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010766:Phosphorylation; D020641:Polymorphism, Single Nucleotide; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D012694:Serine; D048229:Sulfolobus solfataricus / Q000201:enzymology* / Q000235:genetics
D019843:Archaeal Proteins; D012694:Serine