Biomedical Engineering Reference
In-Depth Information
similar to the ACP (acyl carrier protein) domain of fatty acid synthases and
polyketide synthases.
The ACV synthetase is encoded by a large gene without introns (11.5 kb).
This gene was named
pcbAB
(for penicillin and cephalosporin biosynthesis)
and was initially thought to encode two proteins corresponding to a
dipeptide synthetase (PcbA) and a tripeptide synthetase (PcbB), but further
experimental results and cloning of the gene from
P. chrysogenum
(Diez et al.
1990) and
A. chrysogenum
(Gutiérrez et al. 1991) proved that all the tripeptide
biosynthetic steps are catalysed by a single protein (named PcbAB) with a
molecular weight of 414791 (3712 amino acids) in
A. chrysogenum
(Gutiérrez
et al. 1991).
Cyclization of The ACV Tripeptide to Isopenicillin N
The second step, common to penicillin and cephalosporin biosynthesis,
is the cyclization of the linear tripeptide ACV to IPN mediated by the
IPN synthase (also known as ACV cyclase) that converts ACV to IPN, a
hydrophilic penicillin with a L-α-aminoadipyl side chain. The cyclizations
catalysed by the IPN synthase [an iron (Fe
2+
) and ascorbic acid-dependent
dioxygenase] involves the removal of four hydrogen atoms and the
formation of a C-N bond that forms the β-lactam ring and a C-S bond
between C-3 of valine and the SH group of cysteine that gives rise to the
thiozolidine ring of IPN. The enzyme IPN synthase is an intermolecular
dioxygenase that uses molecular oxygen as cosubstrate and ascorbate as
electron donor. This enzyme has been crystallized (Roach et al. 1995) and
the molecular mechanism of electron and proton transfer to the molecular
oxygen has been widely studied (Roach et al. 1997).
Cephalosporin-specifi c Biosynthetic Steps
After IPN formation, the pathway for cephalosporin biosynthesis in
A.
chrysogenum
diverges from that of benzylpenicillin in
P. chrysogenum
.
Whereas the last step of benzylpenicillin biosynthesis involves a
transacylation step in which the α-aminoadipyl side chain is replaced by
a phenylacetic acid moiety, in
A. chrysogenum
and the bacteria producing
cephalosporin-like compounds, IPN is fi rst epimerised to PenN and
then converted by a ring expanding dioxygenase into the cephalosporin
intermediate DAOC. The epimerisation system in fungi is different from
that performed by the bacterial IPN epimerase. Purifi cation of the
A.
chrysogenum
IPN epimerase proved to be diffi cult and unreliable because
the enzyme was extremely labile in cell-free preparations (Baldwin et
al. 1981b, Jayatilake et al. 1981). As a consequence of these facts the
