Biochemical characterization of Bacillus subtilis type II isopentenyl diphosphate isomerase, and phylogenetic distribution of isoprenoid biosynthesis pathways

Abstract

An open reading frame (Acc. no. P50740) on the Bacillus subtilis chromosome extending from bp 184 997-186 043 with similarity to the idi-2 gene of Streptomyces sp. CL190 specifying type II isopentenyl diphosphate isomerase was expressed in a recombinant Escherichia coli strain. The recombinant protein with a subunit mass of 39 kDa was purified to apparent homogeneity by column chromatography. The protein was shown to catalyse the conversion of dimethylallyl diphosphate into isopentenyl diphosphate and vice versa at rates of 0.23 and 0.63 lmolAEmg . NADPH is required under aerobic but not under anaerobic assay conditions. The enzyme is related to a widespread family of (S)-a-hydroxyacid oxidizing enzymes including flavocytochrome b 2 and L-lactate dehydrogenase and was shown to catalyse the formation of [2,[3][4][5][6][7][8][9][10][11][12][13] C 2 ]lactate from [2,[3][4][5][6][7][8][9][10][11][12][13] C 2 ]pyruvate, albeit at a low rate of 1 nmolAEmg. Putative genes specifying type II isopentenyl diphosphate isomerases were found in the genomes of Archaea and of certain eubacteria but not in the genomes of fungi, animals and plants. The analysis of the occurrence of idi-1 and idi-2 genes in conjunction with the mevalonate and nonmevalonate pathway in 283 completed and unfinished prokaryotic genomes revealed 10 different classes. Type II isomerase is essential in some important human pathogens including Staphylococcus aureus and Enterococcus faecalis where it may represent a novel target for anti-infective therapy.

A new family of enzymes catalyzing the first committed step of the methylerythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis in bacteria

“…These studies proposed that the MVA pathway is germane to archaea (where it is required for the production of membrane ether-linked isoprenoid lipids) whereas the MEP pathway is the ancestral route in bacteria, serving for the production of precursors for quinones, carotenoids, and membrane stabilizers such as hopanoids. Although the majority of bacteria exclusively use the MEP pathway for IPP and DMAPP biosynthesis, there are some exceptions (20)(21)(22). For example, several species of parasitic Rickettsia and Mycoplasma bacteria have no genes encoding enzymes of the MVA or the MEP pathway, probably because they obtain their isoprenoids from host cells.…”

Kinetic and Spectroscopic Characterization of Type II Isopentenyl Diphosphate Isomerase from Thermus thermophilus: Evidence for Formation of Substrate-Induced Flavin Species

“…Reduction of the cofactor with NADPH or dithionite was required to generate an active enzyme·reduced flavin complex (15;16;27;29). Laupitz and coworkers (18) reported that B. subtilis type II isomerase purified from an recombinant E. coli strain under anaerobic conditions was active without addition of NADPH to the assay buffer. It is curious to note, that they also reported that added FMN was required for activity in the anaerobic assay and that FMN was not reduced by NADPH in the presence of enzyme.…”

Evidence for the Involvement of Acid/Base Chemistry in the Reaction Catalyzed by the Type II Isopentenyl Diphosphate/Dimethylallyl Diphosphate Isomerase from Staphylococcus aureus

“…The spectral changes for the reduced, enzyme-bound 1-deaza and 5-deazaFMN analogues in the presence of IPP are also consistent with the accumulation of their neutral reduced forms. The protonation in the N1/C1-C2═O2′ region of all three coenzymes may be mediated by Lys186 or His149 (S. aureus numbering), both of which are absolutely conserved among IDI-2 enzymes (15)(16)(17)20). Previously, we have suggested that IPP binding may induce a conformational change that results in a K m value for reduced FMN (200 nM) that is 70-fold lower than the K d (14 µM) value for binding of the reduced FMN to IDI-2 in the absence of IPP (21).…”

“…After shimming and initial peak integration, enzyme was added to the NMR tube, and the appearance of the (Z)-methyl proton resonance of DMAPP (δ = 1.66 ppm) (13,15,28) was followed over 120 min using a Varian Unity 500 MHz NMR spectrometer. The spectra at each individual time point were acquired over a 220 s interval, and a 180 s delay time was used between successive data acquisition periods.…”

“…Since its initial isolation from Streptomyces sp. CL190, IDI-2 enzymes have been found in many eubacteria and archaebacteria as part of either the MVA or the MEP biosynthetic pathways (13)(14)(15). Subsequent biochemical and structural characterization of IDI-2 (13,(15)(16)(17)(18)(19)(20)(21)(22) has revealed that the IDI-2-FMN ox complex is reduced with stoichiometric amounts of NAD(P)H via stereospecific transfer of the pro-S hydride and that the resultant IDI-2-FMN red is capable of performing multiple turnovers (21).…”