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The deazapurine biosynthetic pathway revealed: in vitro enzymatic synthesis of PreQ(0) from guanosine 5'-triphosphate in four steps.

ABSTRACT: Deazapurine-containing secondary metabolites comprise a broad range of structurally diverse nucleoside analogues found throughout biology, including various antibiotics produced by species of Streptomyces bacteria and the hypermodified tRNA bases queuosine and archaeosine. Despite early interest in deazapurines as antibiotic, antiviral, and antineoplastic agents, the biosynthetic route toward deazapurine production has remained largely elusive for more than 40 years. Here we present the first in vitro preparation of the deazapurine base preQ(0), by the successive action of four enzymes. The pathway includes the conversion of the recently identified biosynthetic intermediate, 6-carboxy-5,6,7,8-tetrahydropterin, to a novel intermediate, 7-carboxy-7-deazaguanine (CDG), by an unusual transformation catalyzed by Bacillus subtilis QueE, a member of the radical SAM enzyme superfamily. The carboxylate moiety on CDG is converted subsequently to a nitrile to yield preQ(0) by either B. subtilis QueC or Streptomyces rimosus ToyM in an ATP-dependent reaction, in which ammonia serves as the nitrogen source. The results presented here are consistent with early radiotracer studies on deazapurine biosynthesis and provide a unified pathway for the production of deazapurines in nature.

SUBMITTER: McCarty RM

PROVIDER: S-EPMC2693876 | biostudies-literature | 2009 May

REPOSITORIES: biostudies-literature

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The deazapurine biosynthetic pathway revealed: in vitro enzymatic synthesis of PreQ(0) from guanosine 5'-triphosphate in four steps.

McCarty Reid M RM Somogyi Arpád A Lin Guangxin G Jacobsen Neil E NE Bandarian Vahe V

Biochemistry 20090501 18

Deazapurine-containing secondary metabolites comprise a broad range of structurally diverse nucleoside analogues found throughout biology, including various antibiotics produced by species of Streptomyces bacteria and the hypermodified tRNA bases queuosine and archaeosine. Despite early interest in deazapurines as antibiotic, antiviral, and antineoplastic agents, the biosynthetic route toward deazapurine production has remained largely elusive for more than 40 years. Here we present the first in ...[more]

PMID: 19354300

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Project description:A third of humans carry genetic variants of the ITP pyrophosphatase (ITPase) gene (ITPA) that lead to reduced enzyme activity. Reduced ITPase activity was earlier reported to protect against ribavirin-induced hemolytic anemia and to diminish relapse following ribavirin and interferon therapy for hepatitis C virus (HCV) genotype 2 or 3 infections. While several hypotheses have been put forward to explain the antiviral actions of ribavirin, details regarding the mechanisms of interaction between reduced ITPase activity and ribavirin remain unclear. The in vitro effect of reduced ITPase activity was assessed by means of transfection of hepatocytes (Huh7.5 cells) with a small interfering RNA (siRNA) directed against ITPA or a negative-control siRNA in the presence or absence of ribavirin in an HCV culture system. Low ribavirin concentrations strikingly depleted intracellular GTP levels in HCV-infected hepatocytes whereas higher ribavirin concentrations induced G-to-A and C-to-U single nucleotide substitutions in the HCV genome, with an ensuing reduction of HCV RNA expression and HCV core antigen production. Ribavirin triphosphate (RTP) was dephosphorylated in vitro by recombinant ITPase to a similar extent as ITP, a naturally occurring substrate of ITPase, and reducing ITPA expression in Huh 7.5 cells by siRNA increased intracellular levels of RTP in addition to increasing HCV mutagenesis and reducing progeny virus production. Our results extend the understanding of the biological impact of reduced ITPase activity, demonstrate that RTP is a substrate of ITPase, and may point to personalized ribavirin dosage according to ITPA genotype in addition to novel antiviral strategies.IMPORTANCE This study highlights the multiple modes of action of ribavirin, including depletion of intracellular GTP and increased hepatitis C virus mutagenesis. In cell culture, reduced ITP pyrophosphatase (ITPase) enzyme activity affected the intracellular concentrations of ribavirin triphosphate (RTP) and augmented the impact of ribavirin on the mutation rate and virus production. Additionally, our results imply that RTP, similar to ITP, a naturally occurring substrate of ITPase, is dephosphorylated in vitro by ITPase.

Dataset Information

The deazapurine biosynthetic pathway revealed: in vitro enzymatic synthesis of PreQ(0) from guanosine 5'-triphosphate in four steps.

Publications

The deazapurine biosynthetic pathway revealed: in vitro enzymatic synthesis of PreQ(0) from guanosine 5'-triphosphate in four steps.

Similar Datasets

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