RESUMO
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.
Assuntos
Isomerases de Ligação Dupla Carbono-Carbono/química , Streptococcus pneumoniae/enzimologia , Isomerases de Ligação Dupla Carbono-Carbono/genética , Isomerases de Ligação Dupla Carbono-Carbono/metabolismo , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , Desenho de Fármacos , Hemiterpenos , Humanos , Modelos Moleculares , Infecções Pneumocócicas/microbiologia , Conformação Proteica , Streptococcus pneumoniae/química , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/metabolismoRESUMO
Type-2 isopentenyl diphosphate isomerase (IDI-2) is a key flavoprotein involved in the biosynthesis of isoprenoids. Since fully reduced flavin mononucleotide (FMNH2) is needed for activity, it was decided to crystallize the enzyme under anaerobic conditions in order to understand how this reduced cofactor binds within the active site and interacts with the substrate isopentenyl diphosphate (IPP). In this study, the protein was expressed and purified under aerobic conditions and then reduced and crystallized under anaerobic conditions. Crystals grown by the sitting-drop vapour-diffusion method and then soaked with IPP diffracted to 2.1â Å resolution and belonged to the hexagonal space group P6322, with unit-cell parameters a = b = 133.3, c = 172.9â Å.
