Kinetic and Binding Studies of Streptococcus pneumoniae Type 2 Isopentenyl Diphosphate:Dimethylallyl Diphosphate Isomerase

Abstract

Type 2 isopentenyl diphosphate:dimethylallyl diphosphate isomerase (IDI-2) converts isopentenyl diphosphate (IPP) to dimethylallyl diphosphate (DMAPP), the two fundamental building blocks of isoprenoid molecules. IDI-2 is found in many species of bacteria and is a potential antibacterial target since this isoform is non-homologous to the type 1 enzyme in Homo sapiens. IDI-2 requires a reduced flavin mononucleotide to form the catalytically active ternary complex, IDI-2·FMNH2·IPP. For IDI-2 from the pathogenic bacterium Streptococcus pneumoniae, the flavin can be treated kinetically as a dissociable cosubstrate in incubations with IPP and excess NADH. Under these conditions, the enzyme follows a modified sequential ordered mechanism where FMN adds before IPP. Interestingly, the enzyme shows sigmoidal behavior when incubated with IPP and NADH with varied concentrations of FMN in aerobic conditions. In contrast, sigmoidal behavior is not seen in incubations under anaerobic conditions where FMN is reduced to FMNH2 before the reaction is initiated by addition of IPP. Stopped-flow experiments revealed that FMN, whether bound to IDI-2 or without enzyme in solution, is slowly reduced in a pseudo-first-order reaction upon addition of excess NADH (k(red)(FMN) = 5.7 × 10(-3) s(-1) and k(red)(IDI-2·FMN) = 2.8 × 10(-3) s(-1)), while reduction of the flavin is rapid upon addition of NADH to a mixture of IDI-2·FMN, and IPP (k(red)(IDI-2·FMN·IPP) = 8.9 s(-1)). Similar experiments with dithionite as the reductant gave k(red)(FMN) = 221 s(-1) and k(red)(IDI-2·FMN) = 411 s(-1). Dithionite reduction of FMN in the IDI-2·FMN and IPP mixture was biphasic with k(red)(IDI-2·FMN·IPP (fast)) = 326 s(-1) and k(red)(IDI-2·FMN·IPP (slow)) = 6.9 s(-1) The pseudo-first-order rate constant for the slow component was similar to those for NADH reduction of the flavin in the IDI-2·FMN and IPP mixture and may reflect a rate-limiting conformational change in the enzyme.

The type II isopentenyl Diphosphate:Dimethylallyl diphosphate isomerase (IDI-2): A model for acid/base chemistry in flavoenzyme catalysis

“…These studies have consistently yielded k cat values of approximately 1 s −1 , and K m values for IPP and reduced FMN in the low μM and nM ranges, respectively. Recent work by the Poulter group on the Streptococcus pneumoniae enzyme suggests an ordered sequential mechanism for IDI-2 with reduced FMN binding before IPP [45, 46]. This observation is in line with the observed binding affinities of IPP and FMN, as well as with the relative positioning of FMN and IPP in the IDI-2 active site as determined from the available X-ray crystal structures [48, 49].…”

Enhancement of the catalytic activity of Isopentenyl diphosphate isomerase (IDI) from Saccharomyces cerevisiae through random and site-directed mutagenesis

“…7 d). Due to hydrophobicity of Phe-195 and Cys-256, Ala-106, Phe-107 and Cys-139 could fixed substrate better [ 22 ]. The amino acid substitutions at the position of 195 and 256 enlarged the hydrophobic scope, and the active pocket indirectly therefore enhancing enzymatic activity to some extent.…”

Construction of Functional Monomeric Type 2 Isopentenyl Diphosphate:Dimethylallyl Diphosphate Isomerase

“…UV–visible spectra for m- sp IDI-2 were recorded under anaerobic conditions as described previously . Apo-m- sp IDI-2 was reconstituted with FMN using the procedure described previously except that all washes were performed under ambient O 2 conditions . The resulting m- sp IDI-2·FMN solution, and the substrates IPP and DMAPP were degassed and transferred to an anaerobic chamber.…”

“…IDI-2 requires reduced flavin mononucleotide (FMN) for activity, and several lines of evidence suggest that the isomerization catalyzed by IDI-2 proceeds through a protonation and deprotonation process similar to that of IDI-1. − An X-ray crystal structure of the catalytically active enzyme with bound substrate reveals that the protonation–deprotonation steps must involve proton shuttling between the isoprenoid substrate and the reduced flavin. Although several groups have suggested that a zwitterionic form of FMNH 2 is the acid–base system responsible for protonation and deprotonation, the structure of the catalytically active flavin has not been rigorously established.…”

“…Although several groups have suggested that a zwitterionic form of FMNH 2 is the acid–base system responsible for protonation and deprotonation, the structure of the catalytically active flavin has not been rigorously established. Kinetic and binding studies of sp IDI-2 suggest that binding of IPP causes a conformational change that increases the binding affinity of the enzyme for the flavin cofactor and increases the rate of reduction of FMN to FMNH 2 by NADH . These properties were attributed to a change in the conformation of the homotetramer upon binding of IPP to one of the subunits.…”