Biochemical analysis of the modular enzyme inosine 5'-monophosphate dehydrogenase.
Two prominent domains have been identified in the X-ray crystal structure of inosine-5'-monophosphate dehydrogenase (IMPDH), a core domain consisting of an alpha/beta barrel which contains the active site and an inserted subdomain whose structure is less well defined. The core domain encompassing amino acids 1-108 and 244-514 of wild-type human IMPDH (II) connected by the tetrapeptide linker Ile-Arg-Thr-Gly was expressed. The subdomain including amino acids 99-244 of human wild-type IMPDH (II) was expressed as a His-tagged fusion protein, where the His-tag was removable by enterokinase cleavage. These two proteins as well as wild-type human IMPDH (II), all proteins expressed in Escherichia coli, have been purified to apparent homogeneity. Both the wild-type and core domain proteins are tetrameric and have very similar enzymatic activities. In contrast, the subdomain migrates as a monomer or dimer on a gel filtration column and lacks enzymatic activity. Circular dichroism spectropolarimetry indicates that the core domain retains secondary structure very similar to full-length IMPDH, with 30% alpha-helix and 30% beta-sheet vs 33% alpha-helix and 29% beta-sheet for wild-type protein. Again, the subdomain protein is distinguished from both wild-type and core domain proteins by its content of secondary structure, with only 15% each of alpha-helix and beta-sheet. These studies demonstrate that the core domain of IMPDH expressed separately is both structurally intact and enzymatically active. The availability of the modules of IMPDH will aid in dissecting the architecture of this enzyme of the de novo purine nucleotide biosynthetic pathway, which is an important target for immunosuppressive and antiviral drugs.
Nimmesgern
E
E
Vertex Pharmaceuticals Incorporated, 130 Waverly Street, Cambridge, Massachusetts, 02139-4242, USA. Nimmesgern@portal.vpharm.com
Black
J
J
Futer
O
O
Fulghum
J R
JR
Chambers
S P
SP
Brummel
C L
CL
Raybuck
S A
SA
Sintchak
M D
MD
D000818:Animals; D017123:Bacteriophage T7 / Q000235:genetics; D001665:Binding Sites; D002942:Circular Dichroism; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D006639:Histidine / Q000378:metabolism; D006801:Humans; D007168:IMP Dehydrogenase / Q000737:chemistry*; D007700:Kinetics; D008875:Middle Aged; D015335:Molecular Probes / Q000378:metabolism; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014158:Transcription, Genetic
D015335:Molecular Probes; D011993:Recombinant Fusion Proteins; D006639:Histidine; D007168:IMP Dehydrogenase