Defining the active site of Schizosaccharomyces pombe C-terminal domain phosphatase Fcp1.
Fcp1 is an essential protein serine phosphatase that dephosphorylates the C-terminal domain (CTD) of RNA polymerase II. By testing the effects of serial N- and C-terminal deletions of the 723-amino acid Schizosaccharomyces pombe Fcp1, we defined a minimal phosphatase domain spanning amino acids 156-580. We employed site-directed mutagenesis (introducing 24 mutations at 14 conserved positions) to locate candidate catalytic residues. We found that alanine substitutions for Arg(223), Asp(258), Lys(280), Asp(297), and Asp(298) abrogated the phosphatase activity with either p-nitrophenyl phosphate or CTD-PO(4) as substrates. Structure-activity relationships were determined by introducing conservative substitutions at each essential position. Our results, together with previous mutational studies, highlight a constellation of seven amino acids (Asp(170), Asp(172), Arg(223), Asp(258), Lys(280), Asp(297), and Asp(298)) that are conserved in all Fcp1 orthologs and likely comprise the active site. Five of these residues (Asp(170), Asp(172), Lys(280), Asp(297), and Asp(298)) are conserved at the active site of T4 polynucleotide 3'-phosphatase, suggesting that Fcp1 and T4 phosphatase are structurally and mechanistically related members of the DXD phosphotransferase superfamily.
Hausmann
Stéphane
S
Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.
Shuman
Stewart
S
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D001120:Arginine / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry; D001665:Binding Sites; D020134:Catalytic Domain; D004252:DNA Mutational Analysis; D004305:Dose-Response Relationship, Drug; D017353:Gene Deletion; D008239:Lysine / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009596:Nitrophenols / Q000737:chemistry / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000737:chemistry / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000737:chemistry* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D012568:Schizosaccharomyces / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid
D000596:Amino Acids; D009596:Nitrophenols; D009943:Organophosphorus Compounds; D001224:Aspartic Acid; C008644:nitrophenylphosphate; D001120:Arginine; D010749:Phosphoprotein Phosphatases; C116022:carboxy-terminal domain phosphatase; D008239:Lysine; D000409:Alanine