Purification, kinetic characterization, and site-directed mutagenesis of <em>Methanothermobacter thermautotrophicus</em> RFAP Synthase Produced in <em>Escherichia coli</em>

Matthew E. Bechard; Payam Farahani; Dina Greene; Anna Pham; Andrew Orry; Madeline E. Rasche; Matthew E. Bechard; Payam Farahani; Dina Greene; Anna Pham; Andrew Orry; Madeline E. Rasche

doi:10.3934/microbiol.2019.3.186

AIMS Microbiology

2019, Volume 5, Issue 3: 186-204. doi: 10.3934/microbiol.2019.3.186

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Research article

Purification, kinetic characterization, and site-directed mutagenesis of Methanothermobacter thermautotrophicus RFAP Synthase Produced in Escherichia coli

1.
Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, TN 37232
2.
Chemistry and Biochemistry Department, California State University at Fullerton, 800 North State College Blvd., Fullerton, CA 92834
3.
Northern California Regional Laboratories, The Permanente Medical Group, Berkeley, CA 94710
4.
Molsoft L.L.C., 11199 Sorrento Valley Road, S209, San Diego, CA 92121

Received: 02 May 2019 Accepted: 15 July 2019 Published: 23 July 2019

Methane-producing archaea are among a select group of microorganisms that utilize tetrahydromethanopterin (H₄MPT) as a one-carbon carrier instead of tetrahydrofolate. In H₄MPT biosynthesis, β-ribofuranosylaminobenzene 5’-phosphate (RFAP) synthase catalyzes the production of RFAP, CO₂, and pyrophosphate from p-aminobenzoic acid (pABA) and phosphoribosyl-pyrophosphate (PRPP). In this work, to gain insight into amino acid residues required for substrate binding, RFAP synthase from Methanothermobacter thermautotrophicus was produced in Escherichia coli, and site-directed mutagenesis was used to alter arginine 26 (R26) and aspartic acid 19 (D19), located in a conserved sequence of amino acids resembling the pABA binding site of dihydropteroate synthase. Replacement of R26 with lysine increased the K_M for pABA by an order of magnitude relative to wild-type enzyme without substantially altering the K_M for PRPP. Although replacement of D19 with alanine produced inactive enzyme, asparagine substitution allowed retention of some activity, and the K_M for pABA increased about threefold relative to wild-type enzyme. A molecular model developed by threading RFAP synthase onto the crystal structure of homoserine kinase places R26 in the proposed active site. In the static model, D19 is located close to the active site, yet appears too far away to influence ligand binding directly. This may be indicative of the protein conformational change predicted previously in the Bi-Ter kinetic mechanism and/or formation of the active site at the interface of two subunits. Due to the vital role of RFAP synthase in H₄MPT biosynthesis, insights into the mode of substrate binding and mechanism could be beneficial for developing RFAP synthase inhibitors designed to reduce the production of methane as a greenhouse gas.
- methanogenesis,
- methanopterin,
- RFAP synthase,
- site-directed mutagenesis,
- substrate binding
Citation: Matthew E. Bechard, Payam Farahani, Dina Greene, Anna Pham, Andrew Orry, Madeline E. Rasche. Purification, kinetic characterization, and site-directed mutagenesis of Methanothermobacter thermautotrophicus RFAP Synthase Produced in Escherichia coli[J]. AIMS Microbiology, 2019, 5(3): 186-204. doi: 10.3934/microbiol.2019.3.186

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Abstract

Methane-producing archaea are among a select group of microorganisms that utilize tetrahydromethanopterin (H₄MPT) as a one-carbon carrier instead of tetrahydrofolate. In H₄MPT biosynthesis, β-ribofuranosylaminobenzene 5’-phosphate (RFAP) synthase catalyzes the production of RFAP, CO₂, and pyrophosphate from p-aminobenzoic acid (pABA) and phosphoribosyl-pyrophosphate (PRPP). In this work, to gain insight into amino acid residues required for substrate binding, RFAP synthase from Methanothermobacter thermautotrophicus was produced in Escherichia coli, and site-directed mutagenesis was used to alter arginine 26 (R26) and aspartic acid 19 (D19), located in a conserved sequence of amino acids resembling the pABA binding site of dihydropteroate synthase. Replacement of R26 with lysine increased the K_M for pABA by an order of magnitude relative to wild-type enzyme without substantially altering the K_M for PRPP. Although replacement of D19 with alanine produced inactive enzyme, asparagine substitution allowed retention of some activity, and the K_M for pABA increased about threefold relative to wild-type enzyme. A molecular model developed by threading RFAP synthase onto the crystal structure of homoserine kinase places R26 in the proposed active site. In the static model, D19 is located close to the active site, yet appears too far away to influence ligand binding directly. This may be indicative of the protein conformational change predicted previously in the Bi-Ter kinetic mechanism and/or formation of the active site at the interface of two subunits. Due to the vital role of RFAP synthase in H₄MPT biosynthesis, insights into the mode of substrate binding and mechanism could be beneficial for developing RFAP synthase inhibitors designed to reduce the production of methane as a greenhouse gas.

Abbreviation CFE: Cell-free extract; DTT: dithiothreitol; HMPT: tetrahydromethanopterin MES, 2-(-morpholino) ethanesulfonic acid; ITC: isothermal titration calorimetry; ABA: -aminobenzoic acid; PIPES: piperazine-′-bis (2-ethanesulfonic acid); PRPP: 5-phospho--ribose 1-diphosphate; PRTase: phosphoribosyl transferase; RFAP: ribofuranosylaminobenzene 5′-phosphate; SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis; TES: -[Tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid;
Acknowledgments

This research was supported by National Science Foundation grant numbers MCB-0420766, MCB-1020200, and CHE-1508801. The authors are grateful for the research contributions of Rosemarie Garcia and Alex Nguyen.

Conflict of interest

All authors declare no conflicts of interest in this paper.

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