Identification of an Archaeal type II isopentenyl diphosphate isomerase in methanothermobacter thermautotrophicus

Abstract

Isopentenyl diphosphate (IPP):dimethylallyl diphosphate isomerase catalyzes the interconversion of the fundamental five-carbon homoallylic and allylic diphosphate building blocks required for biosynthesis of isoprenoid compounds. Two different isomerases have been reported. The type I enzyme, first characterized in the late 1950s, is widely distributed in eukaryota and eubacteria. The type II enzyme was recently discovered in Streptomyces sp. strain CL190. Open reading frame 48 (ORF48) in the archaeon Methanothermobacter thermautotrophicus encodes a putative type II IPP isomerase. A plasmid-encoded copy of the ORF complemented IPP isomerase activity in vivo in Salmonella enterica serovar Typhimurium strain RMC29, which contains chromosomal knockouts in the genes for type I IPP isomerase (idi) and 1-deoxy-D-xylulose 5-phosphate (dxs). The dxs gene was interrupted with a synthetic operon containing the Saccharomyces cerevisiae genes erg8, erg12, and erg19 allowing for the conversion of mevalonic acid to IPP by the mevalonate pathway. His6-tagged M. thermautotrophicus type II IPP isomerase was produced in Escherichia coli and purified by Ni2+ chromatography. The purified protein was characterized by matrix-assisted laser desorption ionization mass spectrometry. The enzyme has optimal activity at 70 degrees C and pH 6.5. NADPH, flavin mononucleotide, and Mg2+ are required cofactors. The steady-state kinetic constants for the archaeal type II IPP isomerase from M. thermautotrophicus are as follows: K(m), 64 microM; specific activity, 0.476 micromol mg(-1) min(-1); and k(cat), 1.6 s(-1).

FIG. 1.

MVA (left) and MEP (right) biosynthetic pathways to IPP and DMAPP. Abbreviations: AcCoA, acetyl CoA; AcAcCoA, acetoacetyl-CoA; HMGCoA, 3-hydroxy-3-methylglutaryl-CoA; MVAP, phosphomeualonate; MVAPP, diphosphomenalonate; DXP, deoxyxylulose phosphate; GP, glyceraldehyde phosphate; MEP, methylerythritol phosphate; CDP-MEP, cytidine methylerythritol diphosphate; COP-MEPP, cytidine phosphomethylerythritol diphosphate; cMEPP, cyclomethylerythritol diphosphate; HDMAPP, hydroxydimethylallyl diphosphate.

FIG. 2.

Growth of S. enterica serovar Typhimurium strains JMSB0351, JMSB0354, and RMC29 on selective media.

FIG. 3.

Dependence of type II IPP isomerase activity from M. thermautotrophicus on temperature (A), pH (B), [MgCl₂] (C), [FMN] (D), and [NADPH] (E).

FIG. 4.

Sedimentation equilibrium data for type II IPP isomerase from M. thermautotrophicus. The lower panel shows experimental data points for three different loading concentrations (□, 4.47 μM; ▵, 2.24 μM; ○, 1.12 μM) of the protein. The upper panels show the residuals for fits of experimental data to the tetrameric model. The small and random deviations indicate a good fit corresponding to a K_D of 17 μM.