4-Oxalocrotonate tautomerase, its homologue YwhB, and active vinylpyruvate hydratase: synthesis and evaluation of 2-fluoro substrate analogues.
A series of 2-fluoro-4-alkene and 2-fluoro-4-alkyne substrate analogues were synthesized and examined as potential inhibitors of three enzymes: 4-oxalocrotonate tautomerase (4-OT) and vinylpyruvate hydratase (VPH) from the catechol meta-fission pathway and a closely related 4-OT homologue found in Bacillus subtilis designated YwhB. All of the compounds were potent competitive inhibitors of 4-OT with the monocarboxylated 2E-fluoro-2,4-pentadienoate and the dicarboxylated 2E-fluoro-2-en-4-ynoate being the most potent. Despite the close mechanistic and structural similarities between 4-OT and YwhB, these compounds were significantly less potent inhibitors of YwhB with K(i) values ranging from 5- to 633-fold lower than those determined for 4-OT. The study of VPH is complicated by the fact that the enzyme is only active as a complex with the metal-dependent 4-oxalocrotonate decarboxylase (4-OD), the enzyme following 4-OT in the catechol meta-fission pathway. A structure-based sequence analysis identified 4-OD as a member of the fumarylacetoacetate hydrolase (FAH) superfamily and implicated Glu-109 and Glu-111 as potential metal-binding ligands. Changing these residues to a glutamine verified their importance for enzymatic activity and enabled the production of soluble E109Q4-OD/VPH or E111Q4-OD/VPH complexes, which retained full hydratase activity but had little decarboxylase activity. Subsequent incubation of the E109Q4-OD/VPH complex with the substrate analogues identified the 2E and 2Z isomers of the monocarboxylated 2-fluoropent-2-en-4-ynoate as competitive inhibitors. The combined results set the stage for crystallographic studies of 4-OT, YwhB, and VPH using these inhibitors as ligands.
Division of Medicinal Chemistry, College of Pharmacy, The University of Texas, Austin, Texas 78712-1071, USA.
William H
WH
Johnson
Susan C
SC
Wang
Thanuja M
TM
Stanley
Robert M
RM
Czerwinski
Jeffrey J
JJ
Almrud
Gerrit J
GJ
Poelarends
Alexey G
AG
Murzin
Christian P
CP
Whitman
D000473:Alkanes / Q000737:chemistry* / Q000494:pharmacology; D000480:Alkynes / Q000737:chemistry* / Q000494:pharmacology; D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D001667:Binding, Competitive; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005069:Evaluation Studies as Topic; D005461:Fluorine / Q000737:chemistry / Q000494:pharmacology; D006836:Hydro-Lyases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D007535:Isomerases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D008024:Ligands; D008969:Molecular Sequence Data; D046915:Penicillin-Binding Proteins; D033903:Periplasmic Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D000473:Alkanes; D000480:Alkynes; D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D008024:Ligands; C436969:PbpG protein, Bacillus subtilis; D046915:Penicillin-Binding Proteins; D033903:Periplasmic Proteins; D005461:Fluorine; C110907:2-hydroxypentadienoic acid hydratase; D006836:Hydro-Lyases; C021918:4-oxalocrotonate tautomerase; D007535:Isomerases