title: 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.
authors.affiliation: Department of Chemistry, University of Utah , 315 South 1400 East, Salt Lake City, Utah 84112, United States.
authors.forename: Matthew Walter
authors.initials: MW
authors.lastname: Janczak
authors.affiliation: Department of Chemistry, University of Utah , 315 South 1400 East, Salt Lake City, Utah 84112, United States.
authors.forename: C Dale
authors.initials: CD
authors.lastname: Poulter
mesh_terms: D000332:Aerobiosis; D019748:Carbon-Carbon Double Bond Isomerases / Q000737:chemistry* / Q000378:metabolism*; D018360:Crystallography, X-Ray; D005486:Flavin Mononucleotide / Q000378:metabolism; D005415:Flavins / Q000378:metabolism; D045782:Hemiterpenes / Q000737:chemistry / Q000378:metabolism*; D006873:Hydroquinones / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D009943:Organophosphorus Compounds / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D013056:Spectrophotometry, Ultraviolet; D013296:Streptococcus pneumoniae / Q000201:enzymology* / Q000378:metabolism
chemical_list: D005415:Flavins; D045782:Hemiterpenes; D006873:Hydroquinones; D009943:Organophosphorus Compounds; C540087:flavin mononucleotide hydroquinone; C004809:isopentenyl pyrophosphate; C043060:3,3-dimethylallyl pyrophosphate; D005486:Flavin Mononucleotide; D019748:Carbon-Carbon Double Bond Isomerases; C021453:isopentenyldiphosphate delta-isomerase