Mutational analysis of vaccinia virus nucleoside triphosphate phosphohydrolase I, a DNA-dependent ATPase of the DExH box family.
Vaccinia virus nucleoside triphosphate phosphohydrolase I (NPH-I) is a DNA-dependent ATPase that serves as a transcription termination factor during viral mRNA synthesis. NPH-I is a member of the DExH box family of nucleic acid-dependent nucleoside triphosphatases (NTPases), which is defined by the presence of several conserved sequence motifs. We have assessed the contributions of individual amino acids (underlined) in motifs I (GxGKT), II (DExHN), III (SAT), and VI (QxxGRxxR) to ATP hydrolysis by performing alanine scanning mutagenesis. Significant decrements in ATPase activity resulted from mutations at nine positions: Lys-61 and Thr-62 (motif I); Asp-141, Glu-142, His-144, and Asn-145 (motif II); and Gln-472, Arg-476, and Arg-479 (motif VI). Structure-function relationships at each of these positions were clarified by introducing conservative substitutions and by steady-state kinetic analysis of the mutant enzymes. Comparison of our findings for NPH-I with those of mutational studies of other DExH and DEAD box proteins underscores similarities as well as numerous disparities in structure-activity relationships. We conclude that the functions of the conserved amino acids of the NTPase motifs are context dependent.
Martins
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Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.
Gross
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CH
Shuman
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D017766:Acid Anhydride Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites; D004247:DNA / Q000378:metabolism; D006868:Hydrolysis; D016296:Mutagenesis; D043583:Nucleoside-Triphosphatase; D013329:Structure-Activity Relationship; D014616:Vaccinia virus / Q000201:enzymology* / Q000235:genetics
D000255:Adenosine Triphosphate; D004247:DNA; D017766:Acid Anhydride Hydrolases; D000251:Adenosine Triphosphatases; D043583:Nucleoside-Triphosphatase