Isolation of an intermediate in clavulanic acid biosynthesis

Enzyme Multifunctionality by Control of Substrate Positioning Within the Catalytic Cycle—A QM/MM Study of Clavaminic Acid Synthase

“…Thus, the distance between the C3‐bound pro ‐ R hydrogen of DGPC and the oxo ligand is reasonably short, that is, 2.03 Å, which facilitates the reaction, whereas the pro ‐ S hydrogen atom points away from the ferryl species. For the CAS:DHC complex, the analogous distance for the C3‐bound hydrogen atom is 2.34 Å, which is also in agreement with the labelling studies and suggests the desaturation reaction of DHC starts with C3−H bond cleavage [42] …”

“…For the CAS:DHC complex, the analogous distance for the C3-bound hydrogen atom is 2.34 ,w hich is also in agreement with the labellings tudies and suggestst he desaturation reactiono fD HC starts with C3ÀHb ond cleavage. [42] The investigated enzyme-substrate complex (S)h as aq uintet spin state, which has been shown to be the ground state of analogous ferryl species in numerous experimental [43][44][45] and computational studies. [46][47][48][49][50] In the optimised structure of S,t he Fe IV =Od istance is 1.61 ,i na greement with the value obtained experimentally for taurine/a-ketoglutarate dioxygenase (1.62 ) [51] and also matching the resultso fp reviousc omputa-tional studies.…”

Roles of 2-oxoglutarate oxygenases and isopenicillin N synthase in β-lactam biosynthesis

“…134 – 136 CAS then catalyses two further reactions: first the unusual bicyclisation of proclavaminic acid to form dihydroclavaminic acid, then a desaturation reaction to form clavaminic acid. 137 The subsequent steps leading to clavulanic acid, in which the amino group of (3 S ,5 S )-clavaminic acid is replaced with a hydroxyl group and the two stereocenters are epimerised, have not been fully elucidated. 138 The final step in the pathway involves NADPH/NADH dependent reduction of (3 R ,5 R )-clavaldehyde to give clavulanic acid.…”

The enzymes of β-lactam biosynthesis

“…products resulting upon incubation of PCA and partially puri-ed CAS, which revealed the presence of a minor resonance at d $ 5.4 corresponding to the C-5 proton of 2,8-dihydroclavaminic acid. 415,417 Repeating the experiment with C-3-deuterated PCA enabled the isolation of the saturated bicyclic [ 2 H 2 ]-dihydroclavaminic acid, because of the operation of a primary isotope effect which slows down the exocyclic double bond formation. Upon incubation with CAS, the puried dihydroclavaminic acid was converted to clavaminic acid demonstrating the intermediacy of dihydroclavaminic acid in the cyclisation of PCA to clavaminic acid.…”