Biomedical Engineering Reference
In-Depth Information
α-ketoglutarate-dependent dioxygenase very similar to CefE and CefF
of
S. clavuligerus
and
Amydatopsis lactamdurans
. In these enzymes the
α-ketoglutarate substrate is decarboxylated to succinate and CO
2
using one
of the oxygen atoms of molecular oxygen, whereas the other is incorporated
into the DAOC to form the 3'-OH group of DAC.
The final step in the cephalosporin biosynthetic pathway is the
acetylation of DAC using acetyl-CoA. The enzyme (CefG) that performs
this reaction belongs to the widely distributed acetyltransferase family and
is very closely related to homoserine O-acetyltransferase (Met2), an enzyme
involved in the methionine biosynthetic pathway (Gutiérrez et al. 1992).
The fi logenetic tree of these enzymes show that CefG evolved probably by
gene duplication of
met2
(since there is a strong conservation) or perhaps
by horizontal gene cluster. The substrate specifi city of this enzyme for the
acetyl-CoA is quite specifi c and no natural cephalosporins with C3'-side
chains other than the acetyl group are found in
A. chrysogenum
, although
they occur in cephamycin producing bacteria (Coque et al. 1995). Although
there are several other acetyl- and acyltransferases in
A. chrysogenum
, none of
them is able to replace the CefG, since the blocked mutants in the
cefG
gene
are unable to form cephalosporin C and instead, secrete DAC (Matsuda et
al. 1992, Gutiérrez et al. 1992). Although it was initially reported (Matsuda
et al. 1992) that the native enzyme might be processed into two subunits, a
molecular characterization of this enzyme proved, by inmunodetection, that
the non-processed 50-kDa enzyme encoded by
cefG
is present in the wild
type
A. chrysogenum
(Brotzu's strain) and into two improved cephalosporin
producing strains (Velasco et al. 1999). Edman degradation proved that the
immunodetected CefG protein starts at the expected Met 1 of the ORF. The
crystal structure of the DAC acetyltranferase in complexes with reaction
intermediates has been elucidated. The enzyme was found to belong to the
α/β class of acetyltransferases. As other enzymes of this family, it contains
a catalytic triad (His-Asp-Ser) in its active center. The acetyl transferase
reaction proceeds by sequential transfer of the acetyl group from acetyl-CoA
to the serine in the active site of the enzyme followed by a second transfer
of this group from the acyl-enzyme to DAC (Lejon et al. 2008).
CEPHALOSOPRIN C BIOSYNTHETIC GENES
Like another β-lactam-producing fungi and bacteria, most of the genes
involved in antibiotic biosynthesis are located in clusters (Barredo et al.
1989, Diez et al. 1990, Martin 1992, Keller and Hohn 1997, Gutiérrez et al.
1999). The penicillin and cephamycin biosynthetic genes in
P. chrysogenum
and
Streptomyces
species respectively, are organised into a single cluster
(Martín et al. 2005, Liras and Martín 2006), whereas in
A. chrysogenum
all
