Chemistry Reference
In-Depth Information
the enzymatic machinery. Thus, a directed order of additional reaction steps can
be implemented by controlled translocation, and NRPSs are thus often described
as assembly line-like machineries. The PCP domain consists of 90 amino acid
residues, roughly, and is known to rearrange itself to at least three different
tertiary structures in aqueous solution, as is necessary for interaction with the
surrounding domains at certain stages of synthesis (14). Just like ACPs, the PCP
domains are also dependent on a posttranslational modification to function. This
modification is the attachment of a 4
-phosphopantetheine cofactor to a con-
served serine residue. The terminal thiol group of this cofactor is the nucleophile
that attacks the mixed anhydride (acyl-AMP) and therefore covalently binds the
NRPS substrates
via
a thioester bond. After such an acylation, the PCP domain
directs the substrate toward the next processing domain. If we leave out any
optional modifying domains at this point, this next domain would generally be a
condensation (C) domain.
The C domain is needed for the condensation of two biosynthetic interme-
diates during nonribosomal peptide assembly (15). The PCP-bound electrophilic
donor substrate is presented from the N-terminal side of the synthetase. On the
other side, the nucleophilic acceptor substrate—bound analogously to the PCP
domain of the next module—reaches back to the active site of the C domain
from the other direction (downstream). In the first condensation reaction of an
NRPS, both of these substrates would typically be aminoacyl groups connected
to their PCP domains. Condensation is initiated by the nucleophilic attack of
the
α
-amino group of the acceptor substrate onto the thioester group of the
donor substrate. The cofactor of the upstream PCP domain is released, and the
resulting amide bond now belongs to the dipeptide, which remains bound to
the downstream PCP domain. Thus, a translocation of the condensation prod-
uct toward the next module has occurred. All condensation reactions are strictly
unidirectional—always transporting the growing product chain toward the mod-
ule closer to the C-terminus of the machinery. The elongated peptide then serves
as the donor in a subsequent condensation step on the next module. Usually,
there are as many condensation domains in an NRPS as there are peptide bonds
in the linear peptide product. This general translocation model implies that the
biosynthesis is linear—altogether dependent on delicate, situationally changing
affinities that guarantee correct timing for each reaction and that prevents side
reactions (Fig. 4.4). Even though this model successfully puts the biosynthetic
enzymes in relation with their products for most known NRPS systems, some
exceptions are known: The structures of syringomycin (16) or coelichelin (17)
cannot be sufficiently explained by merely deciphering the buildup of their NRPSs
when using this model. Obviously, other regulatory mechanisms and forms of
inter-domain communication are not yet fully understood.
When the last condensation reaction has occurred, the linear precursor needs
to be released from the enzyme. For this important last step, several mecha-
nisms are known: simple hydrolysis of the thioester (balhimycin, vancomycin),
intramolecular cyclization leading to a lactam (tyrocidine, bacitracin) or a lac-
tone (surfactin), or even reductive thioester cleavage (linear gramicidin). In some
