New role of flavin as a general acid-base catalyst with no redox function in type 2 isopentenyl-diphosphate isomerase.
Using FMN and a reducing agent such as NAD(P)H, type 2 isopentenyl-diphosphate isomerase catalyzes isomerization between isopentenyl diphosphate and dimethylallyl diphosphate, both of which are elemental units for the biosynthesis of highly diverse isoprenoid compounds. Although the flavin cofactor is expected to be integrally involved in catalysis, its exact role remains controversial. Here we report the crystal structures of the substrate-free and complex forms of type 2 isopentenyl-diphosphate isomerase from the thermoacidophilic archaeon Sulfolobus shibatae, not only in the oxidized state but also in the reduced state. Based on the active-site structures of the reduced FMN-substrate-enzyme ternary complexes, which are in the active state, and on the data from site-directed mutagenesis at highly conserved charged or polar amino acid residues around the active site, we demonstrate that only reduced FMN, not amino acid residues, can catalyze proton addition/elimination required for the isomerase reaction. This discovery is the first evidence for this long suspected, but previously unobserved, role of flavins just as a general acid-base catalyst without playing any redox roles, and thereby expands the known functions of these versatile coenzymes.
Unno
Hideaki
H
Department of Applied Chemistry, Faculty of Engineering, Nagasaki University, Bunkyo-machi, Nagasaki, Nagasaki 852-8521, Japan.
Yamashita
Satoshi
S
Ikeda
Yosuke
Y
Sekiguchi
Shin-Ya
SY
Yoshida
Norie
N
Yoshimura
Tohru
T
Kusunoki
Masami
M
Nakayama
Toru
T
Nishino
Tokuzo
T
Hemmi
Hisashi
H
D000143:Acids; D000468:Alkalies; D000595:Amino Acid Sequence; D055162:Biocatalysis; D019748:Carbon-Carbon Double Bond Isomerases / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D005415:Flavins / Q000737:chemistry* / Q000378:metabolism*; D045782:Hemiterpenes; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010084:Oxidation-Reduction; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D013379:Substrate Specificity; D016944:Sulfolobus / Q000201:enzymology
D000143:Acids; D000468:Alkalies; D005415:Flavins; D045782:Hemiterpenes; D008024:Ligands; D019748:Carbon-Carbon Double Bond Isomerases; C021453:isopentenyldiphosphate delta-isomerase