Clavaminate

Scheme 10.30 Part of clavulanic acid biosynthesis. Bonds installed byclavaminic acid synthase (CAS) are circled. CAS clavaminic acid synthase. PAH proclavaminic acid amidino hydrolase.

Other examples of a-keto acid-dependent enzymes are mammalian proline hydroxylase and bacterial clavaminate synthase [113]. The latter enzyme is of particular interest as it is responsible for the catalysis of three individual steps in the biosynthesis of the (3-lactamase inhibitor clavulanic acid (Scheme 10.30). 

A complete cycle of iron-oxo generation and substrate oxidation with the consumption of one equivalent 02 and one equivalent a-ketoglutarate is necessary for each of the reactions catalyzed by clavaminate synthase. The different substrates utilized by clavaminate synthase adopt slightly different positions relative to the... 

Figure 13.21 Mononuclear non-haem iron enzymes from each of the five families in structures which are poised for attack by 02. (a) The extradiol-cleaving catechol dioxygenase, 2,3-dihydroxy-biphenyl 1,2-dioxygenase (b) the Rieske dioxygenase, naphthalene 1,2-dioxygenase (c) isopenicillin N-synthase (d) the ot-ketoglutarate dependent enzyme clavaminate synthase and (e) the pterin-dependent phenylalanine hydroxylase. (From Koehntop et al., 2005. With kind permission of Springer Science and Business Media.)...

The common motif shared by non-heme iron oxygenases contains an active site, where two histidines and one carboxylate occupy one face of the Fe(ll) coordination sphere. These enzymes catalyze a variety of oxidative modification of natural products. For example, in the biosynthesis of clavulanic acid, clavaminic acid synthase demonstrates remarkable versatility by catalyzing hydroxylation, oxidative ring formation and desaturation in the presence of a-ketoglutarate (eq. 1 in Scheme 7.22) [80]. The same theme was seen in the biosynthesis of isopenicillin, the key precursor to penicillin G and cephalosporin, from a linear tripeptide proceeded from a NRPS, where non-heme iron oxygenases catalyze radical cyclization and ring expansion (eq. 2 in Scheme 7.22) [81, 82]. 

Fluorination of vinorelbine was thus performed in super acid medium (HF-SbFs). A super electrophilic agent, such as a chloromethyl or a Br+ cation is generated in s/fu from a chloromethane (CHCI3, CCI4) or from N-Bromo Succinimide (NBS). It is able to abstract a hydrogen from the protonated alkaloid, leading to a cation that can be trapped by an halide anion present in the medium [105,106], Difluorination remarkably occurs selectively at C-4 of the clavamine fragment (Fig. 37) [105]. 

Scheme 2.2 Examples of reactions catalyzed by and RNA by the protein AlkB [54] (R = sugar al

C.). (2000) Structural origins of the selectivity of the trifunctional oxygenase clavaminic add synthase. Nature Structural and Molecular Biology, 7,... 

Figure 10 Several reactions catalyzed by a-keto acid-dependent iron enzymes, (a) prolyl 4-hydroxylase, (b) deacetoxycephalosporin C synthase (DAOCS), (c) 4-hydroxyphenylpyruvate (HPP) dioxygenase and (d) clavaminate synthase 2 (CAS)...

Zhou J, Kelly WL, Bachmmann BO, Gunsior M, Townsend CA, Solomon EL Spectroscopic studies of substrate interactions with clavaminate synthase 2, a multifunctional alpha-KG-dependent non-heme iron enzyme correlation with mechanisms and reactivities. J. Am. Chem. Soc. 2001 123 7388-7398. 

A large family of these enzymes is now known, and their enzymology and structures have been reviewed. A number of crystal structures have been obtained for enzymes in this family, and in each case the mononuclear iron(II) center is coordinated by a His,His,Glu motif, also observed in the extradiol catechol dioxygenases, and in other nonheme iron-dependent enzymes. Structural studies on clavaminic acid synthase have indicated the structural basis for the separate hydroxylation and oxidative cyclization/ desaturation reactions catalyzed by this enzyme. ... 

A number of protein crystal structures has been obtained for this class of enzymes, including those of isopenicillin-N-synthase, " deacetoxycephalosporin C synthase (DAOCS) Figure 14, and clavaminate synthase (CAS) these structures are currently being studied with regards to the effect of structure on the regioselectivity exhibited by these enzymes. For DAOCS the structure shows a jelly-roll motif with the active site situated in a shallow pocket on the edge of... 

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