Synthetase gramicidin

Similarly, iterative NRPSs operate in a linear fashion but utilize at least one domain or module multiple times for the synthesis of a single NRP product. Thus, peptides assembled by iterative synthetases contain short, repeating units of peptide building blocks. In such systems, the terminal PCP-TE (or infrequendy PCP-C) didomain is responsible for both condensation of the repeating peptide units and chain release from the assembly line. NRPs biosynthesized in this manner include enniatin, enterobactin, bacillibactin, " gramicidin and the depsi-peptides valinomycin and cereulide. Of these examples, condensation of the precursor peptides for both enterobactin and gramicidin S has been extensively studied and will be discussed in detail. 

Several characterized NRPSs utilize alternative methods for chain termination. In some synthetases, the TE domain of the final module is replaced by an NAD(P)H-dependent reductase domain. Reduction of a peptidyl-S-PCP substrate through a two-electron reaction leads to the formation of a transient aldehyde, which is subsequently converted into a cyclic imine or hemiaminal through intramolecular cyclization. This two-electron reaction is utilized in the biosynthesis of nostocyclopeptides, the saframycins, ° and anthramycin. Alternatively, a four-electron reduction to the primary alcohol is observed in the biosynthesis of mycobacterial peptidolipids, linear gramicidin," " the myxalamides, lyngbyatoxin, " and myxochelin A 75,76 alternative four-electron reduction pathway involving aldehyde formation, transamination, and reduction to a primary amine occurs in the biosynthesis of myxochelin B. ... 

As noted previously, linear gramicidin" " and anabaenopeptilid" synthetases begin with modules containing formylation (F) domains in an F-A-PCP initiation module. In vitro experiments have revealed that formylation domains act upon aminoacyl-S-PCP intermediates before amide bond formation occurs within the downstream C domain. These F domains likely utilize w °-formyltetrahydrofolate as a cofactor. ... 

An adenylation domain from the gramicidin S synthetase provided the first atomic resolution structural information for an NRPS domain (Figure 11 (a)). The excised protein is part of the first module of the synthetase. The domain is responsible for both activating the amino acid phenylalanine and loading the building block onto the adjacent PCP domain. The structure demonstrated that A domains share a common... 

Early work summarized in Sec. I.B had shown that ACV is made from L-Aad, L-Cys, and L-Val, that 5-(L-a-Aad)-L-Cys (AC) may be converted into ACV, but L-Cys-D-Val is not converted. Likewise, D-Val is not a substrate for ACV synthetase, contrary to the first-characterized NRPS systems of gramicidin S and tyrocidine [2,3], These observations are in agreement with the scheme, except for the incorporation of AC. This dipeptide was later shown to be activated as an adenylate. 

As most NRPS multienzymes are multidomain proteins with multiple activation domains, multiple sites may participate in the reactions assayed, and no clear result concerning a single specific site may result. In ACV synthetases, the nonadditivity of the initial rates has been observed in the S. clavuligerus enzyme [35] and the A. chrysogenum enzyme [1]. Two or more site activations of one substrate amino acid could be expected to depend on different binding constants, and thus be detectable by kinetic analysis. So far, however, no evidence for mixed types of concentration dependence has been found. It is thus not yet clear if nonadditivity results from misactivation or alteration of kinetic properties in the presence of multiple substrates. In the case of gramicidin S synthetase 2, evidence for misactivations has been reported [59],... 

R Kittelberger, M Pavela-Vrancic, El von Dohren. Active site titration of gramicidin S synthetase 2 evidence for misactivation and editing in nonribosomal peptide biosynthesis. FEBS Lett 461 145-148, 1999. 

A Gadow, J Vater, W Schlumbohm, Z Palacz, J Salnikow, H Kleinkauf. Gramicidin S synthetase. Stability of reactive thioester intermediates and formation of 3-amino-2-piperidone. Eur J Biochem 132 229-234, 1983. 

T Stachelhaus, CT Walsh. Mutational analysis of the epimerization domain in the initiation module PheATE of gramicidin S synthetase. Biochemistry 39 5775-5787, 2000. 

W Schlumbohm, T Stein, C Ullrich, J Vater, M Krause, MA Marahiel, V Kruft, B Wittmann-Liebold. An active site serine is involved in covalent substrate amino acid binding at each reaction center of gramicidin S synthetase. J Biol Chem 266 23135-23141, 1991. 

T Stein, J Vater, B Wittmann-Liebold, B Franke, M Panico, R McDowell, HR Morris. Detection of4 -phosphopantetheine at the thioester binding site for L-valine of gramicidin S synthetase. FEBS Lett 340 39 44, 1994. 

The catalytic mechanisms and molecular recognition properties of peptide synthetases have been studied for several decades [169]. Nonribosomal peptides are assembled on a polyenzyme-protein template, first postulated by Lipmann [170]. The polyenzyme model was refined into the thiotemplate mechanism (Fig. 11) in which the amino acid substrates are covalently bound via thioester linkages to active site sulfhydryls of the enzyme and condensed via a processive mechanism involving a 4 -phosphopantetheine carrier [171-173].The presence of a covalently attached pantetheine cofactor was first established in a cell-free system that catalyzed enzymatic synthesis of the decapeptides gramicidin S and tyrocidine. As in the case of fatty acid synthesis, its role in binding and translocating the intermediate peptides was analyzed [174,175]. 

In the case of linear gramicidin, the N-terminus of the nonribosomal peptide carries a formyl group (10). Just like in the bacterial ribosomal synthesis, only a formylated first building block is processed additionally by the corresponding enzymatic machinery. Thus, one can find a distinct formylation (F) domain at the very N-terminus of the synthetase. Another formylated NRPS product is coelichelin whose N-terminal ornithine residue is believed to be Nj-formylated in trans by a formyltransferase genetically associated with the NRPS (17). Formyl-tetrahydrofolate is used as source of the formyl group by these enzymes. 

Luo L, Burkart MD, Stachelhaus T, Walsh CT. Substrate recognition and selection by the initiation module PheATE of gramicidin 31. S synthetase. J. Am. Chem. Soc. 2001 123 11208-11218. 

In most peptides synthetases an epimerization domain that mediates D-amino acid incorporation is embedded within the module. Thus, L-amino acids are substrates of the synthetase and in situ epimerization occurs during peptide chain elongation. The first module of gramicidin S synthetase from Bacillus brevis is a well studied example [39]. This module recognizes L-phenylalanine, and the tightly bound intermediate is epimerized to fhe D-Phe-enzyme com-... 

As described above, the C. purpurea LPS complex is unique among eukaryotes in having its activities divided between two different polypeptides. In its two-polypeptide nature, LPS resembles some prokaryotic peptide synthetases such as tyrocidine synthetase and gramicidin synthetase. However, unlike the prokaryotic peptide synthetases consisting of two polypeptides, the available DNA sequence of cppl (Tudzynski et al., 1999) indicates that its product does not begin with a recognizable condensation domain, as would be typical of the receiving synthetase of a dual-polypeptide system (Marahiel et al., 1997 von Dohren et al., 1997). 

Although no 3D stmcture for any 4CL is currently available, biochemical and 3D gramicidin S synthetase 1 (PheA) structural analyses of adenylate-forming enzymes, such as firefly luciferases, peptide synthetases (e.g., which... 

The 3D structural analyses of firefly luciferase (apo form, 2.0 A resolution), " gramicidin S synthetase 1 (PheA), complexed with AMP (107) and L-Phe (1) (1.9A resolution), " " as well as Acs complexed with adenosine-5 -propylphosphate (109) and CoA (106) (1.75 A resolution), " have established the presence of a two-domain architecture. In each case, a comparatively large N-terminal core domain is connected to a much smaller C-terminal cap domain by a solvated peptide linker, with the active site situated at the interface between the two domains. In addition, 3D structures of CBL " as a binary complex with 4-chlorobenzoyl-adenylate (4-CB-AMP) (111, Figure 23) and as a ternary complex with 4-chlorophenacyl-CoA (113) (aninert... 

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