Characterization of guanine and hypoxanthine phosphoribosyltransferases in Methanococcus voltae

Bowen, Timothy; Lin, Winston; Whitman, William B.

doi:10.1128/jb.178.9.2521-2526.1996

Cited by 15 publications

(15 citation statements)

References 22 publications

(44 reference statements)

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“…These results suggested the presence of nucleotide salvage pathways, which presumably could incorporate base analogs to form toxic nucleotides. The presence of guanine phosphoribosyltransferase and Hpt activities was confirmed in M. voltae, and spontaneous mutants resistant to base analogs lacked these activities (6).…”

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confidence: 85%

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Markerless Mutagenesis inMethanococcus maripaludisDemonstrates Roles for Alanine Dehydrogenase, Alanine Racemase, and Alanine Permease

2005

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Among the archaea, Methanococcus maripaludis has the unusual ability to use L-or D-alanine as a nitrogen source. To understand how this occurs, we tested the roles of three adjacent genes encoding homologs of alanine dehydrogenase, alanine racemase, and alanine permease. To produce mutations in these genes, we devised a method for markerless mutagenesis that builds on previously established genetic tools for M. maripaludis. The technique uses a negative selection strategy that takes advantage of the ability of the M. maripaludis hpt gene encoding hypoxanthine phosphoribosyltransferase to confer sensitivity to the base analog 8-azahypoxanthine. In addition, we developed a negative selection method to stably incorporate constructs into the genome at the site of the upt gene encoding uracil phosphoribosyltransferase. Mutants with in-frame deletion mutations in the genes for alanine dehydrogenase and alanine permease lost the ability to grow on either isomer of alanine, while a mutant with an in-frame deletion mutation in the gene for alanine racemase lost only the ability to grow on D-alanine. The wild-type gene for alanine dehydrogenase, incorporated into the upt site, complemented the alanine dehydrogenase mutation. Hence, the permease is required for the transport of either isomer, the dehydrogenase is specific for the L isomer, and the racemase converts the D isomer to the L isomer. Phylogenetic analysis indicated that all three genes had been acquired by lateral gene transfer from the low-moles-percent G؉C gram-positive bacteria.

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confidence: 85%

“…The previously observed sensitivity of M. maripaludis to 8-azahypoxanthine could be attributed to Hpt encoded by the hpt gene (6,7,14). We constructed a plasmid containing an in-frame deletion in the hpt gene, flanked by 0.9 kb of upstream DNA and 1.4 kb of downstream DNA.…”

Section: Maripaludis Can Use L-or D-alanine As a Nitrogen Sourcementioning

confidence: 99%

Markerless Mutagenesis inMethanococcus maripaludisDemonstrates Roles for Alanine Dehydrogenase, Alanine Racemase, and Alanine Permease

2005

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“…Taken together, these data suggest that HX-containing nucleosides and nucleotides potentially predated guanine-containing nucleotides and nucleosides. This is also supported by the annotated adenine phosphoribosyltransferase (encoded by MJ1655) and its homologues from M. thermautotrophicus and Methanocococcus voltae, which were all demonstrated to be specific for HX and guanine, showing no activity toward adenine (39,40;Miller,unpublished). The presence of these enzymes demonstrates that M. jannaschii, and possibly other methanogens, is unable to use adenine for the salvage of purines and require conversion to HX prior to salvage of the nitrogen-rich purine ring.…”

Section: Discussionmentioning

confidence: 71%

S-Inosyl-l-Homocysteine Hydrolase, a Novel Enzyme Involved inS-Adenosyl-l-Methionine Recycling

Miller

White

2015

J Bacteriol

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S-Adenosyl-L-homocysteine, the product of S-adenosyl-L-methionine (SAM) methyltransferases, is known to be a strong feedback inhibitor of these enzymes. A hydrolase specific for S-adenosyl-L-homocysteine produces L-homocysteine, which is remethylated to methionine and can be used to regenerate SAM. Here, we show that the annotated S-adenosyl-L-homocysteine hydrolase in Methanocaldococcus jannaschii is specific for the hydrolysis and synthesis of S-inosyl-L-homocysteine, not S-adenosyl-L-homocysteine. This is the first report of an enzyme specific for S-inosyl-L-homocysteine. As with S-adenosyl-L-homocysteine hydrolase, which shares greater than 45% sequence identity with the M. jannaschii homologue, the M. jannaschii enzyme was found to copurify with bound NAD ؉ and has K m values of 0.64 ؎ 0.4 mM, 0.0054 ؎ 0.006 mM, and 0.22 ؎ 0.11 mM for inosine, L-homocysteine, and S-inosyl-L-homocysteine, respectively. No enzymatic activity was detected with S-adenosyl-L-homocysteine as the substrate in either the synthesis or hydrolysis direction. These results prompted us to redesignate the M. jannaschii enzyme an S-inosyl-L-homocysteine hydrolase (SIHH). Identification of SIHH demonstrates a modified pathway in this methanogen for the regeneration of SAM from S-adenosyl-L-homocysteine that uses the deamination of S-adenosyl-L-homocysteine to form S-inosyl-L-homocysteine. IMPORTANCEIn strictly anaerobic methanogenic archaea, such as Methanocaldococcus jannaschii, canonical metabolic pathways are often not present, and instead, unique pathways that are deeply rooted on the phylogenetic tree are utilized by the organisms. Here, we discuss the recycling pathway for S-adenosyl-L-homocysteine, produced from S-adenosyl-L-methionine (SAM)-dependent methylation reactions, which uses a hydrolase specific for S-inosyl-L-homocysteine, an uncommon metabolite. Identification of the pathways and the enzymes involved in the unique pathways in the methanogens will provide insight into the biochemical reactions that were occurring when life originated. The recycling of S-adenosyl-L-methionine (SAM)-derived metabolites in Methanocaldococcus jannaschii was recently shown to use a novel enzyme, 5=-deoxyadenosine deaminase (DadD) (1). DadD deaminates three SAM-derived enzymatic products (5=-methylthioadenosine, 5=-deoxyadenosine, and S-adenosyl-L-homocysteine) to produce the inosine analogs ( Fig. 1) (1). The canonical pathway for recycling S-adenosyl-L-homocysteine (SAH) produced from SAM-dependent methyltransferases (2-4) proceeds in a three-step recycling pathway back to SAM (Fig. 2A). SAH is first hydrolyzed to produce adenosine and homocysteine using S-adenosyl-L-homocysteine hydrolase (SAHH). Homocysteine is then methylated by methionine synthase to produce methionine (5). The last step in the pathway is the generation of SAM by combining the adenosyl moiety of ATP with methionine to produce SAM, pyrophosphate, and phosphate (Fig. 2), catalyzed by SAM synthase (6). A similar pathway has been established in M. jannaschii but with ...

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“…The possibility of developing such a system in archaea is supported by several studies of purine and pyrimidine salvage pathways (6,7,17,32,36). In addition to demonstrating the presence of base salvage pathways, it was observed that spontaneous resistance to purine analogs is associated with decreased activity of certain purine salvage enzymes.…”

mentioning

confidence: 66%

“…We suspect that de novo synthesis is repressed in this medium and that the adaptation period required for prototrophic growth involves switching from the salvage pathway back to the de novo pathway. In some methanoarchaea, purine salvage mutants have been isolated by selecting resistance to purine analogs (6,7,17,32,36). Therefore, we tested wild-type M. acetivorans and the hpt1::mini-mar strain for sensitivity to a number of purine analogs (Table 3).…”

Section: Resultsmentioning

confidence: 99%

Development of a Markerless Genetic Exchange Method for Methanosarcina acetivorans C2A and Its Use in Construction of New Genetic Tools for Methanogenic Archaea

Pritchett

Zhang

Metcalf

2004

Appl Environ Microbiol

130

161

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A new genetic technique for constructing mutants of Methanosarcina acetivorans C2A by using hpt as a counterselectable marker was developed. Mutants with lesions in the hpt gene, encoding hypoxanthine phosphoribosyltransferase, were shown to be >35-fold more resistant to the toxic base analog 8-aza-2,6-diaminopurine (8ADP) than was the wild type. Reintroduction of the hpt gene into a ⌬hpt host restored 8ADP sensitivity and provided the basis for a two-step strategy involving plasmid integration and excision for recombination of mutant alleles onto the M. acetivorans chromosome. We have designated this method markerless exchange because, although selectable markers are used during the process, they are removed in the final mutants. Thus, the method can be repeated many times in the same cell line. The method was validated by construction of ⌬proC ⌬hpt mutants, which were recovered at a frequency of 22%. Additionally, a Methanosarcina-Escherichia shuttle vector, encoding the Escherichia coli proC gene as a new selectable marker, was constructed for use in proC hosts. Finally, the markerless exchange method was used to recombine a series of uidA reporter gene fusions into the M. acetivorans proC locus. In vitro assay of ␤-glucuronidase activity in extracts of these recombinants demonstrated, for the first time, the utility of uidA as a reporter gene in Methanosarcina. A >5,000-fold range of promoter activities could be measured by using uidA: the methylcoenzyme M reductase operon fusion displayed ϳ300-fold-higher activity than did the serC gene fusion, which in turn had 16-fold-higher activity than did a fusion to the unknown orf2 gene.

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Characterization of guanine and hypoxanthine phosphoribosyltransferases in Methanococcus voltae

Cited by 15 publications

References 22 publications

Markerless Mutagenesis inMethanococcus maripaludisDemonstrates Roles for Alanine Dehydrogenase, Alanine Racemase, and Alanine Permease

Markerless Mutagenesis inMethanococcus maripaludisDemonstrates Roles for Alanine Dehydrogenase, Alanine Racemase, and Alanine Permease

S-Inosyl-l-Homocysteine Hydrolase, a Novel Enzyme Involved inS-Adenosyl-l-Methionine Recycling

Development of a Markerless Genetic Exchange Method for Methanosarcina acetivorans C2A and Its Use in Construction of New Genetic Tools for Methanogenic Archaea

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