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Application of a Colorimetric Assay to Identify Putative Ribofuranosylaminobenzene 5'-Phosphate Synthase Genes Expressed with Activity in Escherichia coli.

Bechard ME, Chhatwal S, Garcia RE, Rasche ME - Biol Proced Online (2003)

Bottom Line: By lowering the incubation temperature during expression, RFAP synthase from Archaeoglobus fulgidus was produced in E. coli and purified to homogeneity.The production of active RFAP synthase from Methanothermobacter thermautotrophicus was achieved by coexpression of the gene MTH0830 with a molecular chaperone.This is the first direct biochemical identification of a methanogen gene that codes for an active RFAP synthase.

View Article: PubMed Central - HTML - PubMed

Affiliation: Microbiology and Cell Science Department, University of Florida. Gainesville, FL 32611-0700. USA.

ABSTRACT
Tetrahydromethanopterin (H(4)MPT) is a tetrahydrofolate analog originally discovered in methanogenic archaea, but later found in other archaea and bacteria. The extent to which H(4)MPT occurs among living organisms is unknown. The key enzyme which distinguishes the biosynthetic pathways of H(4)MPT and tetrahydrofolate is ribofuranosylaminobenzene 5'-phosphate synthase (RFAP synthase). Given the importance of RFAP synthase in H(4)MPT biosynthesis, the identification of putative RFAP synthase genes and measurement of RFAP synthase activity would provide an indication of the presence of H(4)MPT in untested microorganisms. Investigation of putative archaeal RFAP synthase genes has been hampered by the tendency of the resulting proteins to form inactive inclusion bodies in Escherichia coli. The current work describes a colorimetric assay for measuring RFAP synthase activity, and two modified procedures for expressing recombinant RFAP synthase genes to produce soluble, active enzyme. By lowering the incubation temperature during expression, RFAP synthase from Archaeoglobus fulgidus was produced in E. coli and purified to homogeneity. The production of active RFAP synthase from Methanothermobacter thermautotrophicus was achieved by coexpression of the gene MTH0830 with a molecular chaperone. This is the first direct biochemical identification of a methanogen gene that codes for an active RFAP synthase.

No MeSH data available.


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The reaction of RFAP synthase. Abbreviations: pAB, p-aminobenzoic acid; PRPP, phosphoribosylpyrophosphate; PPi, inorganic pyrophosphate.
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Figure 2: The reaction of RFAP synthase. Abbreviations: pAB, p-aminobenzoic acid; PRPP, phosphoribosylpyrophosphate; PPi, inorganic pyrophosphate.

Mentions: Direct measurement of H4MPT in cells is challenging because the coenzyme is present in low concentrations and is labile to light and oxygen. An alternative approach to investigating H4MPT distribution among organisms is to identify the genes and enzymes involved in H4MPT biosynthesis. One advantage of this approach is that homologs of known H4MPT biosynthesis genes can be identified in the sequenced genomes of various organisms. Putative H4MPT biosynthesis genes can be expressed heterologously, and the activity of the corresponding enzymes can be tested in vitro (11-13). The key enzyme which distinguishes the biosynthetic pathways of H4MPT and H4F is ribofuranosylaminobenzene 5'-phosphate synthase (RFAP synthase) (14). This enzyme catalyzes the reaction between phosphoribosylpyrophosphate and p-aminobenzoic acid (pAB) to form RFAP, carbon dioxide, and inorganic pyrophosphate (Fig. 2) (12, 14, 15). This decarboxylation reaction results in the defining structural difference between H4MPT and H4F, which is the absence in H4MPT of the carbonyl group derived from pAB (Fig. 1) (3). Due to the uniqueness of RFAP synthase as a H4MPT biosynthetic enzyme, the identification of an active RFAP synthase in different organisms could give strong evidence that they contain H4MPT.


Application of a Colorimetric Assay to Identify Putative Ribofuranosylaminobenzene 5'-Phosphate Synthase Genes Expressed with Activity in Escherichia coli.

Bechard ME, Chhatwal S, Garcia RE, Rasche ME - Biol Proced Online (2003)

The reaction of RFAP synthase. Abbreviations: pAB, p-aminobenzoic acid; PRPP, phosphoribosylpyrophosphate; PPi, inorganic pyrophosphate.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC152576&req=5

Figure 2: The reaction of RFAP synthase. Abbreviations: pAB, p-aminobenzoic acid; PRPP, phosphoribosylpyrophosphate; PPi, inorganic pyrophosphate.
Mentions: Direct measurement of H4MPT in cells is challenging because the coenzyme is present in low concentrations and is labile to light and oxygen. An alternative approach to investigating H4MPT distribution among organisms is to identify the genes and enzymes involved in H4MPT biosynthesis. One advantage of this approach is that homologs of known H4MPT biosynthesis genes can be identified in the sequenced genomes of various organisms. Putative H4MPT biosynthesis genes can be expressed heterologously, and the activity of the corresponding enzymes can be tested in vitro (11-13). The key enzyme which distinguishes the biosynthetic pathways of H4MPT and H4F is ribofuranosylaminobenzene 5'-phosphate synthase (RFAP synthase) (14). This enzyme catalyzes the reaction between phosphoribosylpyrophosphate and p-aminobenzoic acid (pAB) to form RFAP, carbon dioxide, and inorganic pyrophosphate (Fig. 2) (12, 14, 15). This decarboxylation reaction results in the defining structural difference between H4MPT and H4F, which is the absence in H4MPT of the carbonyl group derived from pAB (Fig. 1) (3). Due to the uniqueness of RFAP synthase as a H4MPT biosynthetic enzyme, the identification of an active RFAP synthase in different organisms could give strong evidence that they contain H4MPT.

Bottom Line: By lowering the incubation temperature during expression, RFAP synthase from Archaeoglobus fulgidus was produced in E. coli and purified to homogeneity.The production of active RFAP synthase from Methanothermobacter thermautotrophicus was achieved by coexpression of the gene MTH0830 with a molecular chaperone.This is the first direct biochemical identification of a methanogen gene that codes for an active RFAP synthase.

View Article: PubMed Central - HTML - PubMed

Affiliation: Microbiology and Cell Science Department, University of Florida. Gainesville, FL 32611-0700. USA.

ABSTRACT
Tetrahydromethanopterin (H(4)MPT) is a tetrahydrofolate analog originally discovered in methanogenic archaea, but later found in other archaea and bacteria. The extent to which H(4)MPT occurs among living organisms is unknown. The key enzyme which distinguishes the biosynthetic pathways of H(4)MPT and tetrahydrofolate is ribofuranosylaminobenzene 5'-phosphate synthase (RFAP synthase). Given the importance of RFAP synthase in H(4)MPT biosynthesis, the identification of putative RFAP synthase genes and measurement of RFAP synthase activity would provide an indication of the presence of H(4)MPT in untested microorganisms. Investigation of putative archaeal RFAP synthase genes has been hampered by the tendency of the resulting proteins to form inactive inclusion bodies in Escherichia coli. The current work describes a colorimetric assay for measuring RFAP synthase activity, and two modified procedures for expressing recombinant RFAP synthase genes to produce soluble, active enzyme. By lowering the incubation temperature during expression, RFAP synthase from Archaeoglobus fulgidus was produced in E. coli and purified to homogeneity. The production of active RFAP synthase from Methanothermobacter thermautotrophicus was achieved by coexpression of the gene MTH0830 with a molecular chaperone. This is the first direct biochemical identification of a methanogen gene that codes for an active RFAP synthase.

No MeSH data available.


Related in: MedlinePlus