Mutational analysis of baculovirus phosphatase identifies structural residues important for triphosphatase activity in vitro and in vivo.
Baculovirus phosphatase (BVP) and mammalian capping enzyme (Mce1) are members of the RNA triphosphatase branch of the cysteine phosphatase superfamily. Although RNA triphosphatases have a core alpha/beta fold similar to other cysteine phosphatases, there is little conservation of primary structure outside of the cysteine-containing P-loop motif, HCxxxxxR(S/T), that comprises the active site. However, there is extensive primary structure conservation between members of the RNA triphosphatase branch, whether from cellular or viral sources and whether they are bifunctional capping enzymes such as Mce1 or monofunctional RNA phosphatases such as BVP. To evaluate the functional significance of such sequence conservation, we performed a mutational analysis of 14 residues of BVP. We identified three side chains (Trp6, Lys25, and Arg153) as essential for triphosphatase activity in vitro, i.e., W6A, K25A, and R153A were <0.1% as active as wild-type BVP, and were unable to complement a yeast RNA triphosphatase null mutant in vivo. Six other BVP residues (Thr62, Tyr67, Tyr68, Lys82, Glu158, and Arg159) were deemed functionally important, i.e., Ala mutations reduced triphosphatase activity to <20% of wild-type. On the basis of the locations of the equivalent amino acids in the Mce1 crystal structure, we surmise that the essential/important BVP residues ensure proper conformation of the catalytic P-loop (e.g., Arg153 and Tyr68) or other elements of the tertiary structure. Our results highlight a conserved Trp6-Lys25 pi-cation pair essential for BVP function.
Martins
Alexandra
A
Molecular Biology Program, Sloan-Kettering Institute, 1275 York Avenue, New York, New York 10021, USA.
Shuman
Stewart
S
D017766:Acid Anhydride Hydrolases / Q000737:chemistry* / Q000235:genetics*; D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics*; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics; D016367:Baculoviridae / Q000201:enzymology* / Q000235:genetics*; D017124:Conserved Sequence / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods; D004789:Enzyme Activation / Q000235:genetics; D005973:Glutamine / Q000235:genetics; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry* / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D000098556:mRNA Guanylyltransferases
D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D005973:Glutamine; D001224:Aspartic Acid; D009713:Nucleotidyltransferases; D000098556:mRNA Guanylyltransferases; C417245:baculovirus phosphatase; D010744:Phosphoric Monoester Hydrolases; D017766:Acid Anhydride Hydrolases; C023090:RNA triphosphatase; D000409:Alanine