Mechanism of Phosphoanhydride Cleavage by Baculovirus Phosphatase

Martins, Alexandra; Shuman, Stewart

doi:10.1074/jbc.m005748200

Cited by 25 publications

(37 citation statements)

References 39 publications

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“…43 Mutation of this histidine to alanine in the Yersinia PTPase and the baculovirus NTPase/RTPase reduced k cat by two to three orders of magnitude. 43, 53 In At1g05000, the distance between the side chains of His 149 and Cys 150 are compatible with the possible hydrogen bonding.…”

Section: Discussionmentioning

confidence: 85%

Structural and functional characterization of a novel phosphatase from the Arabidopsis thaliana gene locus At1g05000

et al. 2008

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The crystal structure of the protein product of the gene locus At1g05000, a hypothetical protein from A. thaliana, was determined by the multiple-wavelength anomalous diffraction method and was refined to an R factor of 20.4% (Rfree = 24.9%) at 3.3 Å. The protein adopts the α/β fold found in cysteine phosphatases, a superfamily of phosphatases that possess a catalytic cysteine and form a covalent thiol-phosphate intermediate during the catalytic cycle. In At1g05000, the analogous cysteine (Cys150) is located at the bottom of a positively-charged pocket formed by residues that include the conserved arginine (Arg156) of the signature active site motif, HCxxGxxRT. Of 74 model phosphatase substrates tested, purified recombinant At1g05000 showed highest activity toward polyphosphate (poly-P12–13) and deoxyribo- and ribonucleoside triphosphates, and less activity toward phosphoenolpyruvate, phosphotyrosine, phosphotyrosine-containing peptides, and phosphatidyl inositols. Divalent metal cations were not required for activity and had little effect on the reaction.

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Section: Discussionmentioning

confidence: 85%

Structural and functional characterization of a novel phosphatase from the Arabidopsis thaliana gene locus At1g05000

et al. 2008

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“…It is extremely unlikely that the poxvirus, ASFV, or baculovirus capping enzymes are derived from the capping apparatus of their metazoan host cells. Although all metazoan organisms examined to date do encode a bifunctional capping enzyme with an N-terminal RTP domain linked in cis to a Cterminal guanylyltransferase domain (9,20,28,32), the metazoan RTPs are members of the cysteine phosphatase superfamily of metal-independent phosphohydrolases and are completely divergent in both structure and mechanism from the fungal/viral family of metal-dependent triphosphatases (18,19,30). Moreover, there are no discernible homologues of the fungal/viral RTPs in available metazoan proteomes.…”

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confidence: 99%

RNA Triphosphatase Component of the mRNA Capping Apparatus of Paramecium bursaria Chlorella Virus 1

Gong

Shuman

2001

J Virol

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Paramecium bursaria chlorella virus 1 (PBCV-1) elicits a lytic infection of its unicellular green alga host. The 330-kbp viral genome has been sequenced, yet little is known about how viral mRNAs are synthesized and processed. PBCV-1 encodes its own mRNA guanylyltransferase, which catalyzes the addition of GMP to the 5 diphosphate end of RNA to form a GpppN cap structure. Here we report that PBCV-1 encodes a separate RNA triphosphatase (RTP) that catalyzes the initial step in cap synthesis: hydrolysis of the ␥-phosphate of triphosphate-terminated RNA to generate an RNA diphosphate end. We exploit a yeast-based genetic system to show that Chlorella virus RTP can function as a cap-forming enzyme in vivo. The 193-amino-acid Chlorella virus RTP is the smallest member of a family of metal-dependent phosphohydrolases that includes the RNA triphosphatases of fungi and other large eukaryotic DNA viruses (poxviruses, African swine fever virus, and baculoviruses). Chlorella virus RTP is more similar in structure to the yeast RNA triphosphatases than to the enzymes of metazoan DNA viruses. Indeed, PBCV-1 is unique among DNA viruses in that the triphosphatase and guanylyltransferase steps of cap formation are catalyzed by separate viral enzymes instead of a single viral polypeptide with multiple catalytic domains.The m7GpppN cap structure of eukaryotic mRNA is formed cotranscriptionally by three enzymatic reactions: (i) the 5Ј triphosphate end of the nascent RNA is hydrolyzed to a diphosphate by RNA triphosphatase (RTP), (ii) the diphosphate end is capped with GMP by GTP:RNA guanylyltransferase, and (iii) the GpppN cap is methylated by S-adenosylmethionine: RNA (guanine-N7) methyltransferase (27). DNA viruses have evolved diverse capping strategies. The mRNAs of papovaviruses, parvoviruses, adenoviruses, and herpesviruses are transcribed in the nucleus by RNA polymerase II (Pol II), and their 5Ј ends are modified by the host cell's capping and methylating enzymes. However, vaccinia virus and other poxviruses, which replicate in the cytoplasm, encode and encapsidate with the virus particle a multisubunit RNA polymerase and a complete mRNA capping apparatus (26). African swine fever virus (ASFV), which has a cytoplasmic replication phase, also encodes and encapsidates an RNA polymerase and mRNA capping enzymes (24). Baculoviruses, which replicate in the nucleus of insect cells, use Pol II to transcribe early genes, then switch at later times to a virus-encoded transcription system that includes an RNA polymerase and two cap-forming activities-RTP and RNA guanylyltransferase (4,5,14). Paramecium bursaria chlorella virus 1 (PBCV-1) encodes an RNA guanylyltransferase (7), but it is not clear whether it encodes an RNA polymerase and additional mRNA-processing activities.The triphosphatase, guanylyltransferase, and methyltransferase components of the capping apparatus are organized differently in these DNA virus systems. The triphosphatase, guanylyltransferase, and methyltransferase active sites of the vaccinia virus capping enzym...

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“…3B). The phosphate contact to the His-121 side chains is essential, insofar as alanine substitution abolished triphosphatase function in vitro and in vivo and conservative changes to glutamine or asparagine did not restore activity (10). The hydrogen bond of Asn-124 N-␦ to the phosphate is also essential; mutations N124A, N124D, and N124Q abolished BVP triphosphatase activity in vivo and in vitro (10).…”

Section: Resultsmentioning

confidence: 99%

“…Among the RNA-specific members of the cysteine phosphatase family, there is no conserved residue on this loop that could serve as a general acid. Indeed, cleavage of phosphoanhydrides by the RNA-specific cysteine phosphatases does not depend on a general acid catalyst because of the low pK a of the leaving group (3,10). Nonetheless, the structural alignment of Mce1 and BVP revealed that the mobile loop contains a polar residue, such as Gln-88 in BVP and His-94 in Mce1, that might be involved in substrate recognition or binding (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Indeed, although alanine changes at these positions are deleterious, conservative mutational analysis indicates that any polar contacts made by Tyr-67 or Thr-120 to the site 2 phosphate are not functionally important, i.e. Y67F and T120V mutants retain wild-type triphosphatase activity in vitro and in vivo (10,11). It is conceivable that Thr-120 and Tyr-67 help form a pocket for the leaving group phosphate, without needing to interact directly with that phosphate.…”

Section: Discussionmentioning

confidence: 99%

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Crystal Structure of Baculovirus RNA Triphosphatase Complexed with Phosphate

Changela

Martins

Shuman

et al. 2005

Journal of Biological Chemistry

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Baculovirus RNA 5 -triphosphatase (BVP) exemplifies a family of RNA-specific cysteine phosphatases that includes the RNA triphosphatase domains of metazoan and plant mRNA capping enzymes. Here we report the crystal structure of BVP in a phosphatebound state at 1.5 Å resolution. BVP adopts the characteristic cysteine-phosphatase ␣/␤ fold and binds two phosphate ions in the active site region, one of which is proposed to mimic the phosphate of the product complex after hydrolysis of the covalent phosphoenzyme intermediate. The crystal structure highlights the role of backbone amides and side chains of the P-loop motif 118 HCTHGXNRT 126 in binding the cleavable phosphate and stabilizing the transition state. Comparison of the BVP structure to the apoenzyme of mammalian RNA triphosphatase reveals a concerted movement of the Arg-125 side chain (to engage the phosphate directly) and closure of an associated surface loop over the phosphate in the active site. The structure highlights a direct catalytic role of Asn-124, which is the signature P-loop residue of the RNA triphosphatase family and a likely determinant of the specificity of BVP for hydrolysis of phosphoanhydride linkages. mRNA 5Ј cap formation is initiated by hydrolysis of the ␥-phosphate of 5Ј-triphosphate-terminated pre-mRNA. The resulting 5Ј-diphosphate end is then capped by transfer of GMP from GTP to form an inverted terminal dinucleotide structure, G(5Ј)ppp(5Ј)N. The first reaction is catalyzed by RNA 5Ј-triphosphatase and the second by GTP:RNA guanylyltransferase (reviewed in Refs. 1 and 2). In metazoans and plants, the triphosphatase and guanylyltransferase activities reside in a single polypeptide composed of an N-terminal triphosphatase domain fused to a C-terminal guanylyltransferase domain. The metazoan and plant RNA triphosphatases belong to the cysteine phosphatase superfamily (3), which includes phosphoprotein phosphatases (4) and phosphoinositide phosphatases (5).Cysteine phosphatase-type RNA triphosphatases act via a twostep mechanism entailing attack by a cysteine thiolate nucleophile of the enzyme on the ␥-phosphate of triphosphate-terminated RNA to form a cysteinyl-phosphoenzyme intermediate, which is then hydrolyzed to liberate inorganic phosphate. The active site cysteine is located within a signature P-loop motif, HCTHGXNRT.We reported previously (3) the crystal structure of the RNA triphosphatase domain of mouse capping enzyme Mce1 at 1.65 Å resolution. Mce1 adopts a globular fold consisting of a five-stranded parallel ␤ sheet flanked by ␣ helices on both sides. The active site cysteine is located at the bottom of a deep, positively charged pocket formed by essential amino acids that line the pocket walls and surface rim. Structural, biochemical, and mutational results showed that despite sharing a HCXXXXXR(S/T) P-loop motif, a phosphoenzyme intermediate, and a core ␣/␤ fold with other cysteine phosphatases, the mechanism of phosphoanhydride cleavage by Mce1 and its baculovirus homologue BVP 1 differs from that used by phosphoprotein ...

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Mechanism of Phosphoanhydride Cleavage by Baculovirus Phosphatase

Cited by 25 publications

References 39 publications

Structural and functional characterization of a novel phosphatase from the Arabidopsis thaliana gene locus At1g05000

Structural and functional characterization of a novel phosphatase from the Arabidopsis thaliana gene locus At1g05000

RNA Triphosphatase Component of the mRNA Capping Apparatus of Paramecium bursaria Chlorella Virus 1

Crystal Structure of Baculovirus RNA Triphosphatase Complexed with Phosphate

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