Synthase isopenicillin

Fe-dependent oxidase/oxygenases cephalosporin synthase, isopenicillin N-synthase (IPNS) ... 

Fig. 5. Biosynthesis of penicillins when ACV is aminoadipoly cysteinyl valine and IPNS is isopenicillin N synthase and CgH CH2COSCoA represents benzyl coenzyme A. ACV synthetase is thought to catalyze the first step of this reaction sequence.

The key enzyme in this sequence, isopenicillin N synthase (IPNS), has been purified from E. coli (59) and the recombinant enzyme shown to be a single polypeptide of 336 amino acids containing two cysteines, numbers 106 and 255 from the /V-teiminus, and probably a ferrous ion in a nonheme environment. The enzyme has been crystallized and studies undertaken to obtain suitably sized crystals for diffraction studies. 

The tripeptide LLD-ACV is then cydised to isopenicillin N by an oxidative reaction involving the removal of four protons. The enzyme that catalyses this reaction is isopenidllin N synthetase or synthase (IPNS). 

Monooxygenase, Isopenicillin N synthase, Glutathione peroxidase, Methylmalonyl-CoA mutase, PLP-dependent b-lyase... 

The present chapter reviews applications in biocatalysis of the ONIOM method. The focus is on studies performed in our research group, in most cases using the two-layer ONIOM(QM MM) approach as implemented in Gaussian [23], The studied systems include methane monooxygenase (MMO), ribonucleotide reductase (RNR) [24, 25], isopenicillin N synthase (IPNS) [26], mammalian Glutathione peroxidase (GPx) [27,28], Bi2-dependent methylmalonyl-CoA mutase [29] and PLP-dependent P-lyase [30], These systems will be described in more detail in the following sections. ONIOM applications to enzymatic systems performed by other research groups will be only briefly described. 

Oxygen activation is a central theme in biochemistry and is performed by a wide range of different iron and copper enzymes. In addition to our studies of the dinuclear non-heme iron enzymes MMO and RNR, we also studied oxygen activation in the mononuclear non-heme iron enzyme isopenicillin N synthase (IPNS). This enzyme uses O2 to transform its substrate ACV to the penicillin precursor isopenicillin N [53], a key step in the synthesis of the important P-lactam antibiotics penicillins and cephalosporins [54, 55],... 

Figure 2-6. Real and model system used in ONIOM calculations of the reaction mechanism in isopenicillin N synthase...

When looking at the reaction mechanisms of glutathione peroxidase and isopenicillin N synthase, we did not find any reaction step where the transition state is significantly stabilized by long-range electrostatic interactions (i.e. electrostatic interactions outside the active-site model). However, it is should be added that most transition states have been calculated using ONIOM-ME. 

Dioxygen reduction (oxidase activity) and activation for incorporation into organic substrates are catalysed by a number of mononuclear non-haem iron enzymes. We will first consider the intramolecular dioxygenases, in which both atoms of oxygen are introduced into the substrate, then the monoxygenases (in which we choose to include the pterin-dependent hydroxylases), the large family of a-hetoacid-dependent enzymes, and finally isopenicillin N-synthase. 

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 1. Cyclisation of L-a-aminoadipoyl-L-cysteinyl-D-valine (ACV) to isopenicillin N (IPN) by the enzyme isopenicillin N synthase (IPNS).

Fig. 1. Active site of isopenicillin N synthase (IPNS) derived from Aspergillus nidulans turn over of Fe(II) bound ACV substrate (PDB-Code IBKO) (29) in the crystal to Fe(II) bound IPN product (PDB-Code IQJE) (21).

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]. 

Other non-heme enzymes that use dioxygen are 4-methoxy-benzoate O-demethylase, extradiol catechol dioxygenases, the oxidoreductase isopenicillin N synthase, and a-keto acid-dependent enzymes (28). Moreover, the BH4-dependent glyceryl-ether monooxygenase (GEM) also appears to be dependent on nonheme iron for catalysis (see also Section I.E). 

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