Determination of kinetics and the crystal structure of a novel type 2 isopentenyl diphosphate: dimethylallyl diphosphate isomerase from Streptococcus pneumoniae

Abstract

Isopentenyl diphosphate isomerase (IDI) is a key enzyme in the isoprenoid biosynthetic pathway and is required for all organisms that synthesize isoprenoid metabolites from mevalonate. Type 1 IDI (IDI-1) is a metalloprotein that is found in eukaryotes, whereas the type 2 isoform (IDI-2) is a flavoenzyme found in bacteria that is completely absent from human. IDI-2 from the pathogenic bacterium Streptococcus pneumoniae was recombinantly expressed in Escherichia coli. Steady-state kinetic studies of the enzyme indicated that FMNH2 (KM =0.3 μM) bound before isopentenyl diphosphate (KM =40 μM) in an ordered binding mechanism. An X-ray crystal structure at 1.4 Å resolution was obtained for the holoenzyme in the closed conformation with a reduced flavin cofactor and two sulfate ions in the active site. These results helped to further approach the enzymatic mechanism of IDI-2 and, thus, open new possibilities for the rational design of antibacterial compounds against sequence-similar and structure-related pathogens such as Enterococcus faecalis or Staphylococcus aureus.

Keywords: X-ray structures; bisubstrates; flavoproteins; isoprenoids; steady-state kinetics.

Figure 1

Bisubstrate kinetic plots for sp-IDI-2. Reciprocal plots (A and B) and standard plots (C and D) data points are the average from three experiments along with globally fit regression lines.

Figure 2

A) Tetrameric arrangement of the enzyme. FMN cofactor is represented in purple. Insert: Yellow crystals grown after 7 days. B) Cartoon representation of the typical α8β8 TIM-barrel structure of IDI-2 (N-terminus blue to C-terminus red). One end of the barrel is occupied by the reduced flavin cofactor the other is closed by two antiparallel β-sheets. The N-terminal α-helix caps the active site pocket. C) View of the binding pocket. The 2Fo-Fc electron density map (blue) is drawn at 1.0σ.

Figure 3

Superposition of ss-IDI-2 in complex with IPP and FMNH₂ (purple) and sp-IDI-2 in complex with reduced cofactor (green) and sulfate ions (stick and balls). These ions are stabilized by ionic salt bridges with guanine from R5, and by interactions with amide moiety of Q149 and imidazole ring of H9. Interestingly, they lie at the same positions as the C2-atom and the β-phosphate within IPP observed in ss-IDI-2•IPP structure inside the binding pocket (sp numbering/ss numbering).

Figure 4

Surface representation of the active site pocket. Arg5, Lys6, Ser92 and Met151 can be targeted by competitive inhibitors to restrict access to the binding pocket. N-terminal segment is represented in blue and cap the active site in the “closed” conformation of the enzyme.

Scheme 1

Currently proposed mechanisms for IDI-1 (red) and IDI-2 (blue). In the latter case the N5 nitrogen of FMN is likely candidate for the catalytic nucleophile.