Biomedical Engineering Reference
In-Depth Information
epimerization process remained unclear for decades. In 2002 Ullán and
co-workers (2002b) reported that the epimerization reaction is similar to
epimerization systems involved in degradation of rare fatty acids or toxic
metabolites in other eukaryotic cells (Knihinicki et al. 1991, Shieh and Chen
1993, Schmitz et al. 1995) and entirely different from the known pyridoxal
phosphate-dependent epimerase involved in the biosynthesis of bacterial
β-lactam antibiotics (Jensen et al. 1983, Láiz et al. 1990, Martín et al. 2004).
The fungal IPN epimerization system appears to consist of two enzymes
(Ullán et al. 2002b). The fi rst enzyme activates IPN to isopenicillinyl
N-CoA (encoded by
cefD1
) and the second enzyme catalyzes the authentic
epimerization reaction (encoded by
cefD2
) converting isopenicillinyl
N-CoA to penicillinyl N-CoA. The activating CoA is fi nally removed by a
thioesterase (reviewed by Martín et al. 2004).
LATE CEPHALOSPORIN STEPS: CONVERSION OF
PENICILLIN N TO CEPHALOSPORIN C
In
A. chrysogenum
, PenN is converted to DAOC by expansion of the fi ve-
membered thiazolidine ring to give the six-membered dihydrothiazine
ring of DAOC. This reaction is catalyzed by the ring expanding activity
(expandase) of the bifunctional DAOC synthetase/hydroxylase. The
bifunctional enzyme was stimulated by reducing agents (dithiothreitol or
glutathione), ascorbate and ATP. From the molecular point of view, this
enzyme is a dioxygenase that requires α-ketoglutarate, Fe
2+
and molecular
oxygen (Dotzlaf and Yeh 1987, Kupka et al. 1983).
Structural studies of DAOC synthetase revealed that in the fi rst half-
reaction, O
2
and α-ketoglutarate form a reactive iron-oxygen species that
subsequently reacts with PenN to give DAOC (Valegård K et al. 2004).
Many strains of
A. chrysogenum
accumulate PenN suggesting that the
ring expanding step may be limiting for cepahalosporin production in
these strains (Scheidegger et al. 1984, Dotzlaf and Yeh 1987). Indeed, the
production of PeN/IPN may be considered as a complete pathway itself,
independently of the conversion of PenN to cephalosporin C. These
penicillins are well secreted to the culture medium in
A. chrysogenum
cultures.
In the following step the exocyclic CH3 of DAOC is hydroxylated
to form DAC. This hydroxylation step in
A. chrysogenum
is catalysed by
the second of the two related activities of the CefEF protein (expandase/
hydroxylase). In β-lactam producing bacteria, the CefE (expandase)
and CefF (3'-hydroxylase) proteins are similar enzymes (59% identity
in
S. clavuligerus
) that apparently were formed by gene duplication and
specialization. In
A. chrysogenum
, the bifunctional CefEF protein is an
