title: 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.
authors.affiliation: Department of Chemistry, University of Utah, 315 South 1400 East RM 2020, Salt Lake City, Utah 84112 (USA); Department of Chemistry, UNamur, 61 rue de Bruxelles, 5000 Namur (Belgium).
authors.forename: Jerome
authors.initials: J
authors.lastname: de Ruyck
authors.forename: Matthew W
authors.initials: MW
authors.lastname: Janczak
authors.forename: Syam Sundar
authors.initials: SS
authors.lastname: Neti
authors.forename: Steven C
authors.initials: SC
authors.lastname: Rothman
authors.forename: Heidi L
authors.initials: HL
authors.lastname: Schubert
authors.forename: Rita M
authors.initials: RM
authors.lastname: Cornish
authors.forename: Andre
authors.initials: A
authors.lastname: Matagne
authors.forename: Johan
authors.initials: J
authors.lastname: Wouters
authors.forename: C Dale
authors.initials: CD
authors.lastname: Poulter
mesh_terms: D019748:Carbon-Carbon Double Bond Isomerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D015195:Drug Design; D045782:Hemiterpenes; D006801:Humans; D008958:Models, Molecular; D011008:Pneumococcal Infections / Q000382:microbiology; D011487:Protein Conformation; D013296:Streptococcus pneumoniae / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism
chemical_list: D045782:Hemiterpenes; D019748:Carbon-Carbon Double Bond Isomerases; C021453:isopentenyldiphosphate delta-isomerase
keywords: X-ray structures; bisubstrates; flavoproteins; isoprenoids; steady-state kinetics