Biochemistry. 2016 Mar 22. [Epub ahead of print]

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

Janczak MW, Poulter CD.

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 nonhomologous to the type 1 enzyme in H. 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 co-substrate 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 (kredFMN = 5.7 x 10-3 s-1 and kredIDI-2·FMN = 2.8 x 10-3 s-1), while reduction of the flavin is rapid upon addition of NADH to a mixture of IDI-2•FMN, and IPP (kredIDI-2·FMN·IPP = 8.9 s-1). Similar experiments with dithionite as the reductant, gave kredFMN = 221 s-1 and kredIDI-2·FMN = 411 s-1. Dithionite reduction of FMN in the IDI-2•FMN and IPP mixture was biphasic with kredIDI-2·FMN·IPP (fast) = 326 s-1 and kredIDI-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.

PMID: 27003727 [PubMed - as supplied by publisher]