Gramicidin S synthetase

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... 

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. 

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-... 

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... 

The carboxylic acid analogues lacking the a-amino group are not activated by gramicidin S-synthetase. Hydrocinnamic acid, the analogue of phenylalanine, inhibits reaction 1 non-competitively. This compound most likely does not bind to the reaction center of GS 2 (2). Cyclopentane carboxylic acid and isocapronic acid show a very weak inhibition of the L-Pro resp. L-Leu activation. Carboxylic acid analogues of L-Orn (5-aminovaleric acid as well as 4-aminobutyric acid and 6-aminocapronic acid with a shortened or extended chain) do not appreciably affect reaction 4. 

Esters of the substrate amino acids with alcohol residues of appreciable size are accepted by gramicidin S-synthetase and catalyze biosynthesis of gramicidin S (5). The activation mechanism for these compounds still needs to be elucidated. It is interesting that these findings are in contrast to the features observed for the ami noacyl adenylate activation of amino acids by tRNA synthetases in protein biosynthesis which are inhibited by substrate esters. From these results we infer that as in the tRNA synthetases the a-amino group of the substrate amino acids of gramicidin S synthetase is essential for their binding to the aminoacyl adenylate activation sites of the... 

J. Vater, and H. Kleinkauf, A further characterization of phenylalanine racemase, the light enzyme of gramicidin S-synthetase, Biochim. Biophys. Acta 429, 1062 (1976). 

A. L. Demain, Microbiological studies on the formation of gramicidin S synthetases, Biotechnol. Bioeng. 17, 129 (1975). 

The NDL synthetase reaction was shown to require ATP, Mg, and MTL and to be heat labile. When ( Clpropylhygric acid replaced [ C]propylprolinc in the reaction mixture, there was no incorporation of the label into lincomycin. The pH and temperature optima were determined to be 8.0 and 30 C, respectively. When the crude extract was diluted below a certain critical concentration, the activity diminished dramatically. Enzyme activity was also lost after passing the preparation through a size-exclusion column, but the activity could be recovered by combining specific fractions (51). These studies supported the hypothesis that the nature of the NDL synthetase activity was that of an enzyme complex of readily dissociable, nonidentical subunits. The activity of one of the subunits was identified by a propylproline-dependent PP -ATP exchange assay based on the method used for the tyrocidine synthetase subunit assay (50,52). The peptide antibiotic synthetases of tyrocidine synthetase, edeine synthetase, and gramicidin S synthetase have previously been reported as complexes of readily dissociable, nonidentical subunits (52-54). 

Zimmer T-L. Laland SG. Gramicidin S synthetase. Hash, JH, ed. Methods in Enzymology. Vol. 43. Antibiotics. New York Academic Press, 1975 567-579. 

Stein T. Vater J. Krugt V, Wictmann-Liebold B, Fianke P, Panico M. McDowell R, Moms HR. Detection of 4 -phosphopanietheine at the chioester binding sice for i-valine of gramicidin S synthetase 2. FEBS Lett 1994 340 39-44-... 

Tokito K, Hori K, Kurotsu T, Kanda M, Saito Y. Effect of single base substitution at glycine-870 codon of gramicidin S synthetase 2 gene on pioline activation. J Biochem 1993 ... 

Stein T, Kluge B, Barer ], Franke P, Otto A, Wittmann-Licbold B. Gramicidin S synthetase I (phenylalanine racemase). a prototype of amino acid racemascs eomaining the ccrfactor 4 -pKosphopantetheine, Biochem 1995 34 4633-4642. 

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