Clavaminate synthase

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.)...

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

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 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... 

The best Studied enzyme in the pathway is clavaminate synthase (CAS), which is involved in the conversion of prodavaminic acid to clavaminic acid (see Figure 8). CAS oxidatively catalyzes the formation of the oxazolidine ring, which is characteristic of all clavams, including clavulanic acid. Initially, CAS activity was studied in the clavulanic acid producer, S. ciavtdigems (141,142), but recently CAS activity has also been detected... 

Jane J W, an LA, Townsend CA. Purilication and characterization of clavaminate synthase from Sneptomyces onabioocus. J Biol Chem 1995 270 5399-5404. 

Marsh EN, Chang MDT, Townsend CA. Two isozymes of clavaminate synthase central to clavulanic acid formation Cloning and sequencing of both genes from Streptomyces clainJigerus. Biochem 1992 31 12648-12657. 

Paradkar AS, Jensen 5E. Functional analysis of the gene encoding the clavaminate synthase 2 isoenzyme involved in clavulanic acid biosynthesis in Streptomyces davtdjgerus. J Bactennl... 

Lipoxygenase Catechol dioxygenases a-Ketoglutarate-dependent dioxygenases (Clavaminate synthase) Arene dioxygenases... 

Clavaminate synthase is one of the enzymes involved in the biosynthesis of the p-lactamase inhibitor clavulanic acid. The key function of the enzyme is conversion of the monocyclic p-lactam, proclavaminic acid, to the bicyclic clavaminic acid. This process requires ferrous ion as co-factor and a-ketoglutarate (a-KG) and molecular oxygen as cosubstrate. The enzyme has been purified from Streptomyces clavuligerus, Mj- 47 kDa [340] and Streptomyces antibioticus [341]. Binding of histidine and cysteine to iron is suggested [342]. In this case one atom of dioxygen is incorporated into succinate rather than to form hydroxylation products. The mechanism has been studied recently [343-345], and Scheme 20, which involve Fe =0 as an active species, has been proposed... 

Scheme 20. Reaction scheme for oxygenation of proclavamic acid by clavaminate synthase. a-KG and succ denote a-ketoglutarate and succunic acid, respectively [344].

Zhang ZH, Ren JS, Harlos K, McKinnon CH, Clifton IJ, Schofield CJ. 2002. Crystal structure of a clavaminate synthase-Fe(II)-2-oxoglutarate-substrate-NO complex evidence for metal centered rearrangements. FEBS Lett 517(l-3) 7-12. 

Simple examples include the preparation of monomethyl [2- H]succinate (29) for enzyme mechanism studies (Figure 3.13). The carboxyl, C2-dianion was prepared with LDA, freed of all volatiles, then treated with CFsCOO H prepared from 0.44 molar equivalents (vs 29) of tritiated water, thus utilizing the entire quantity of tritium without dilution. Likewise, the 7V-acetylazetidinone 30 (previously deuterated at C3 by a different exchange procedure) was treated with LiHMDS to selectively form the anion at the acetate C2 position, which was then quenched by addition of tritiated water . Compound 30 was used as an intermediate for the preparation of a substrate for studies of the enzyme clavaminate synthase. 

---