Expression of the
            <i>Methanobacterium thermoautotrophicum hpt</i>
            Gene, Encoding Hypoxanthine (Guanine) Phosphoribosyltransferase, in
            <i>Escherichia coli</i>

Sauer, Jørgen; Nygaard, Per

doi:10.1128/jb.181.6.1958-1962.1999

Cited by 12 publications

(5 citation statements)

References 35 publications

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“…Hpt exhibited a linear dependence of product formation up to 20 min at 70 ° C. The M. jannaschii Hpt demonstrates approximately equal specificity ( k cat / K M ) for either hypoxanthine or guanine (Table ). Interestingly, the M. jannaschii Hpt shares low sequence identity (<50%) with the characterized Hpt from M. thermautrophicum and M. voltae and even less with bacterial homologues (<20% identity).…”

Section: Resultsmentioning

confidence: 97%

“…Purine incorporation and metabolism has been evaluated in three methanogens: Methanobacterium thermautotrophicus, Methanococcus vannielii, and Methanococcus voltae . M. thermautotrophicus showed low levels of Ade activity and an active Hpt, M. vannielii demonstrated use of adenine as the sole nitrogen source, and M. voltae was able to incorporate the purines into nucleic acids and demonstrated to have both hypoxanthine and guanine phosphoribosyltransferase activity . The source of adenine in M. jannaschii remains unknown, but one possible source could be from exogenous adenine that is taken up by the cell and then salvaged.…”

Section: Resultsmentioning

confidence: 99%

“…). Hpt has been described in M. thermoautotrophicum and Methanococcus voltae to be specific for hypoxanthine and guanine . Characterizations of Ade and Hpt from M. jannaschii , encoded by the MJ1459 and MJ1655 gene products, respectively, are described here.…”

Section: Introductionmentioning

confidence: 92%

See 2 more Smart Citations

Purine salvage in Methanocaldococcus jannaschii: Elucidating the role of a conserved cysteine in adenine deaminase

Miller

Bevan

et al. 2016

Proteins

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Adenine deaminases (Ade) and hypoxanthine/guanine phosphoribosyltransferases (Hpt) are widely distributed enzymes involved in purine salvage. Characterization of the previously uncharacterized Ade (MJ1459 gene product) and Hpt (MJ1655 gene product) are discussed here and provide insight into purine salvage in Methanocaldococcus jannaschii. Ade was demonstrated to use either Fe(II) and/or Mn(II) as the catalytic metal. Hpt demonstrated no detectable activity with adenine, but was equally specific for hypoxanthine and guanine with a kcat /KM of 3.2 × 10(7) and 3.0 × 10(7) s(- 1) M(- 1) , respectively. These results demonstrate that hypoxanthine and IMP are the central metabolites in purine salvage in M. jannaschii for AMP and GMP production. A conserved cysteine (C127, M. jannaschii numbering) was examined due to its high conservation in bacterial and archaeal homologues. To assess the role of this highly conserved cysteine in M. jannaschii Ade, site-directed mutagenesis was performed. It was determined that mutation to serine (C127S) completely abolished Ade activity and mutation to alanine (C127A) exhibited 10-fold decrease in kcat over the wild type Ade. To further investigate the role of C127, detailed molecular docking and dynamics studies were performed and revealed adenine was unable to properly orient in the active site in the C127A and C127S Ade model structures due to distinct differences in active site conformation and rotation of D261. Together this work illuminates purine salvage in M. jannaschii and the critical role of a cysteine residue in maintaining active site conformation of Ade. Proteins 2016; 84:828-840. © 2016 Wiley Periodicals, Inc.

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Section: Resultsmentioning

confidence: 97%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Purine salvage in Methanocaldococcus jannaschii: Elucidating the role of a conserved cysteine in adenine deaminase

Miller

Bevan

et al. 2016

Proteins

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“…So far, it has often been pointed out and also shown that the insu⁄cient expression of archaeal AT-rich genes in E. coli might be due to the frequent use of the codons, such as AGA and AGG for arginine and AUA for isoleucine, being rarely used in E. coli [11,16,17]. It is known that such rare codons particularly cause problems when the codons are arranged in clusters or are close to each other.…”

Section: Discussionmentioning

confidence: 99%

Overexpression in Escherichia coli of the AT-rich trpA and trpB genes from the hyperthermophilic archaeon Pyrococcus furiosus

Ishida

2002

FEMS Microbiology Letters

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Expression of AT-rich genes from microorganisms such as archaea is often inefficient in Escherichia coli. The trpA and trpB genes encoding the tryptophan synthase subunits were cloned from the hyperthermophilic archaeon Pyrococcus furiosus. No apparent difference in codon bias was found between the genes. However, using a conventional cloning vector having the lac promoter, the trpB gene was expressed poorly in E. coli, whereas the trpA gene was overexpressed. The expression of the trpB gene was remarkably enhanced ( s 12-fold) by the introduction of an overlapping leader open reading frame. The expression of the trpA gene was also improved (V1.5-fold). This approach may be useful for overexpressing various kinds of AT-rich genes. ß

show abstract

“…So far, it has often been pointed out and also shown that the insufficient expression of archaeal AT‐rich genes in E. coli might be due to the frequent use of the codons, such as AGA and AGG for arginine and AUA for isoleucine, being rarely used in E. coli [11,16,17]. It is known that such rare codons particularly cause problems when the codons are arranged in clusters or are close to each other.…”

Section: Discussionmentioning

confidence: 99%

Overexpression inEscherichia coliof the AT-richtrpAandtrpBgenes from the hyperthermophilic archaeonPyrococcus furiosus

Ishida

Oshima

2002

FEMS Microbiology Letters

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Expression of AT-rich genes from microorganisms such as archaea is often inefficient in Escherichia coli. The trpA and trpB genes encoding the tryptophan synthase subunits were cloned from the hyperthermophilic archaeon Pyrococcus furiosus. No apparent difference in codon bias was found between the genes. However, using a conventional cloning vector having the lac promoter, the trpB gene was expressed poorly in E. coli, whereas the trpA gene was overexpressed. The expression of the trpB gene was remarkably enhanced (>12-fold) by the introduction of an overlapping leader open reading frame. The expression of the trpA gene was also improved ( approximately 1.5-fold). This approach may be useful for overexpressing various kinds of AT-rich genes.

show abstract

Expression of the Methanobacterium thermoautotrophicum hpt Gene, Encoding Hypoxanthine (Guanine) Phosphoribosyltransferase, in Escherichia coli

Cited by 12 publications

References 35 publications

Purine salvage in Methanocaldococcus jannaschii: Elucidating the role of a conserved cysteine in adenine deaminase

Purine salvage in Methanocaldococcus jannaschii: Elucidating the role of a conserved cysteine in adenine deaminase

Overexpression in Escherichia coli of the AT-rich trpA and trpB genes from the hyperthermophilic archaeon Pyrococcus furiosus

Overexpression inEscherichia coliof the AT-richtrpAandtrpBgenes from the hyperthermophilic archaeonPyrococcus furiosus

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