The domain order of mammalian capping enzyme can be inverted and baculovirus phosphatase can function in cap formation in vivo Journal Article
| Authors: | Martins, A.; Shuman, S. |
| Article Title: | The domain order of mammalian capping enzyme can be inverted and baculovirus phosphatase can function in cap formation in vivo |
| Abstract: | The bifunctional mammalian mRNA capping enzyme (Mce1) consists of an N-terminal triphosphatase domain Mce1(1-210) fused to a C-terminal guanylyltransferase domain Mce1(211-597). The physical domain order H2N-triphosphatase-guanylyltransferase-COOH mimics the temporal order of the capping reactions. To determine if the physical domain order is functionally important in vivo, we engineered an "inverted" mammalian capping enzyme InvMce1 [H2N-Mce1(211-597)-(1-210)-COOH]. We found that InvMce1 complemented the growth of Saccharomyces cerevisiae cet1Δ and ceg1Δ strains in which the endogenous yeast triphosphatase and guanylyltransferase genes were deleted. By testing truncated versions of InvMce1, we determined that Mce1(1-178) comprises a minimal functional triphosphatase domain. Baculovirus phosphatase (BVP) is a monofunctional single-domain protein with RNA triphosphatase and RNA diphosphatase activities and an undefined role in viral RNA metabolism. Here we demonstrated that BVP can function as an RNA triphosphatase for cap formation in vivo when fused to the C-terminus of Mce1(211-597). By characterizing a series of InvMce1-BVP derivatives with amino acid substitutions in the phosphate-binding loop of BVP, we showed that the in vivo activity of the mutant chimeras in cap formation is contingent upon in vitro phosphohydrolase activity of the respective BVP proteins. BVP catalysis in vitro was not limited to 5′-phosphorylated RNA or nucleotide substrates, but also embraced tripolyphosphatase and pyrophosphatase activities. BVP-specific activities with nucleotide and inorganic substrates were as follows: ATP (14 min-1), ADP (31 min-1), PPPi (3.7 min-1), and PPi (1 min-1). BVP did not hydrolyze AMP. We surmise that BVP has adapted the cysteinyl phosphatase fold to the hydrolysis of phosphoanhydrides. © 2002 Elsevier Science (USA). |
| Keywords: | controlled study; unclassified drug; gene deletion; mutation; nonhuman; binding affinity; protein domain; protein function; protein analysis; mammalia; amino acid substitution; phosphatase; carboxy terminal sequence; protein; in vivo study; in vitro study; triphosphatase; adenosine diphosphate; rna triphosphatase; enzyme activity; acid anhydride hydrolases; chimera; rna; dna; amino acid sequence; molecular sequence data; enzyme analysis; saccharomyces cerevisiae; rna caps; mammal; substrate specificity; amino acid; catalysis; adenosine triphosphate; multienzyme complexes; phosphate; hydrolysis; phosphoric monoester hydrolases; rna metabolism; fungus growth; nucleotide; enzyme; adenosine phosphate; nucleotidyltransferases; inorganic compound; transferase; guanylyltransferase; baculovirus; acid anhydride; phosphoanhydride; unidentified baculovirus; priority journal; article; capping phenomenon; cysteinyl phosphatase; rna diphosphatase |
| Journal Title: | Virology |
| Volume: | 304 |
| Issue: | 2 |
| ISSN: | 0042-6822 |
| Publisher: | Elsevier Inc. |
| Date Published: | 2002-12-20 |
| Start Page: | 167 |
| End Page: | 175 |
| Language: | English |
| DOI: | 10.1006/viro.2002.1606 |
| PUBMED: | 12504559 |
| PROVIDER: | scopus |
| DOI/URL: | |
| Notes: | Export Date: 14 November 2014 -- Source: Scopus |
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