Biomedical Engineering Reference
In-Depth Information
and cephalosporin C have been reviewed elsewhere (Aharonowitz et
al. 1992, Martín et al. 2010) and only a brief overview is made here to
understand the gene-enzyme correlations.
Both the different penicillins and cephalosporin C derive from three
precursor amino acids; L-α-aminoadipic acid, L-cysteine and L-valine
(reviewed in Martín 2000). These three amino acids are activated and
condensed by the action of the enzyme α-aminoadipyl-cysteinyl-valine
synthetase (ACV synthetase), a non-ribosomal peptide sinthetase (NPRS)
that forms the tripeptide δ-(L-α-aminoadipyl-L-cysteinyl-D-valine (ACV).
The ACV synthetase sequentially activates the three substrate amino acids
with ATP to form aminoacyl-adenylates, binds them to the enzyme as
thioesters, epimerizes the L-valine to D-valine and binds together the three
amino acids to form the tripeptide ACV. Finally, the tripeptide (ACV) is
released from the enzyme by the action of an internal thioesterase domain
of the ACV synthetase complex (Zhang and Demain 1992, Aharonowitz et
al. 1993, Martín 2000).
The NPRS contains three repeated modules with the domain
confi guration ATC
.
ATC
.
ATE
.
R, where A stands for amino acid-activating
domains, T are the thiolation domains (which contain a phosphopantetheine
bound to a conserved serine residue in each of the modules) and
C corresponds to the condensation domains that form the peptide
bonds following the activation of the amino acids. The third module
lacks a condensation domain but instead contains an epimerase domain
(E) that is involved in the conversion of L-valine to D-valine while
the nascent tripeptide is still attached to the ACV synthetase. Finally,
in the C-terminal region of the ACV synthetase there is a release (R)
domain that corresponds to an integrated thioesterase activity. The ACV
synthetase of
A. chrysogenum
was purifi ed by Baldwin and co-workers
(1991a), who showed that it contains 3-phosphopantetheine bound to the
enzyme (holoenzyme). This enzyme is very similar to that of
Penicillium
chrysogenum
(Theilgaard et al. 1997) and
Aspergillus nidulans
(van Liempt
et al. 1989). The structure of this complex multienzyme has received
considerable attention (Byford et al. 1997, Kallow et al. 2000) and has
been reviewed in several articles (Aharonowitz et al. 1993, Zhang and
Demain 1992, Martín 2000).
The ACV synthetase apoenzyme is activated by phosphopantetheinylation
catalysed by the enzyme phosphopantetheinyl transferase (PPTase). The
ppt
gene encoding the PPTase has been cloned and characterized in
P.
chrysogenum
(García-Estrada et al. 2008)
but not in
A. chrysogenum
.
The phosphopantetheine moiety is linked to a serine residue that is
conserved in the T sites of the three repeated modules of the ACV synthetase.
This domain, also named PCP (peptidyl carrier protein sequence), is
