Tyrocidine synthetase 1 (TY1), produced by Bacillus brevis ATCC 8185, consists of a single multifunctional polypeptide chain catalyzing the activation, thioesterification, and epimerization of phenylalanine. Because we were concerned about possible posttranslational issues, a comparative study between the wild-type isolate and the in Escherichia coli overexpressed protein was performed. Analysis by matrix assisted laser desorption mass spectrometry (MALDI) provided a molecular mass of 122,516 +/- 120 Da for the recombinant protein, which is in agreement with the value of 122,590 Da calculated from the gene sequence. MALDI analysis of the tryptic fragments revealed that in the recombinant TY1 the putative 4'-phosphopantetheine binding site (562Ser) is not modified by the cofactor. The substrate specificity profiles of the amino acid dependent ATP[32P]PPi exchange reactions were identical, including activation of L-phenylserine, L-tyrosine, and L-methionine. However, the rates of the reverse adenylation reaction for the recombinant protein were only 22% relative to those of the wild-type enzyme. The aminoacylation levels of about 60% for TY1 from Bacillus brevis reduced to 1.4% in the overexpressed protein. A similar distribution of the D- and the L-isomer was detected at the thioester attachment site. The pI values of the wild-type and expressed TY1 are 4.9 and 5.0, respectively. In conclusion, it could be established that apo- and holo-TY1 differ in their amino acid activating properties. Posttranslational modification by 4'-phosphopantetheine is an essential requirement for aminoacylation, epimerization, and thus the functioning of the multienzyme in peptide synthesis.

Peptide synthetases consist of linearly arranged catalytic units, which by sequence alignment show equally spaced amino-acid-activating segments/modules of 600-700 amino acid residues. The consensus sequence comprises a new class of sequence motifs which are shared by some carboxylactivating enzymes, but which do not occur in aminoacyl-tRNA synthetases. The catalytic properties of peptide synthetases with respect to the nucleotide substrate were investigated by enzyme kinetic studies. In the activation reaction ATP may be substituted by 2'-deoxy-ATP (dATP) and 7-deazaadenosine 5'-triphosphate, substrate analogues which are not recognised by many aminoacyl-tRNA synthetases, and may thus prove useful alternative substrates in the detection of peptide synthetases within complex protein mixtures. ATP derivatives substituted at C2 are substrates, while those substituted at C8 are not, indicating a preference for the anti-conformation in substrate binding. Kinetic studies revealed that coenzyme A is a non-competitive inhibitor of the activation reaction, suggesting the presence of a second nucleotide binding site which accommodates nucleotides with phosphate in the C2' or C3' position. This substrate and inhibition profile is markedly different from that of aminoacyl-tRNA synthetases and indicative of a separate homogeneous family of carboxylactivating enzymes.Amino acids can be incorporated into peptides by two peptide-forming systems, the ribosomal system and the nonribosomal multienzymic system. In the ribosomal system, amino acids are activated by aminoacyl-tRNA synthetases as tRNA esters and peptide bond formation is directed by the ribosome. In the nonribosomal system, amino acids are activated on multienzymes also directing peptide bond formation. We looked for possible common features in the primary structure between the two amino-acid-activating systems. In both cases, amino acids are activated by the energy derived from hydrolysis of an ATP a-P linkage.Linear and cyclic low-molecular-mass peptides, containing non-protein constituents like hydroxy and D-amino acids, are usually produced by peptide synthetases, multienzymes employing the thiotemplate mechanism [l 1. These enzymes range in molecular mass between 123 -1400 kDa [2]. So far, a number of peptide synthetases have been purified and characterised and several amino acid sequences have been published [3 -211. Analysis of the primary structures shows that they are organised in highly conserved and repeated functional units. These structural features support the proposed mechanism whereby amino acids activated as aminoacyladenylates at different specific sites on the multienzyme are subsequently linked into the peptide chain [22]. The Abbreviations. ACV, &(L-a-aminoadipyl)-L-cysteinyl-D-valine;RTP, purineriboside S'-triphosphate; 7-deazaATP, 7-deazaadenosine 5'-triphosphate. Surprisingly, even though peptide synthetases and aminoacyl-tRNA synthetases catalyse similar reactions, there are no significant sequence similarities. Therefore, if the peptide t...

Sequence analysis of peptide synthetases revealed extensive structure similarity with firefly luciferase, whose crystal structure has recently become available, providing evidence for the localization of the active site at the interface between two subdomains separated by a distorted linker region [Conti, E., Franks, N. P. & Brick, P. (1996) Structure 4, 287-2981. The functional importance of two flexible loops, corresponding to the linker region of firefly luciferase and the highly conserved (S/T)GT(T/S)GXPKG core sequence, has been studied in view of the proposed conformational changes by the use of mutant analysis, limited proteolysis and chemical modification of tyrocidine synthetase 1. Substitution of the highly conserved Arg416, residing in the loop separating the subdomains of the adenylation domain, resulted in profound loss of activity. Limited proteolysis of the mutant suggested significant structural changes as manifested by lack of protection to degradation in the presence of substrates, revealing a probable disturbance of the induced-fit mechanism regulating the transformation from an open to a closed conformation. Mutants, obtained by replacement of the conserved Lys186 from the (S/T)GT(T/S)GXPKG core sequence, displayed only minor differences in substrate-binding affinity despite significant reduction of catalytic efficiency. Residue Lys186 appears to play an important role in either stabilization of the bound substrate through charge-charge-interactions, and/or fixing of the loop for maintainance of the active-site conformation.

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Characterization of the nucleotide binding domain in peptide synthetases was approached by photoaffinity labeling of tyrocidine synthetase 1 (TY1) with 2-azidoadenosine triphosphate (2-azido-ATP). Exposure of TY1 in the presence of photolabel to irradiation with ultraviolet light resulted in a time-dependent covalent modification of the enzyme with a concomitant loss of catalytic activity. Inactivation was not observed if incubation was performed in the absence of either light or the nucleotide analogue. Specificity of labeling was indicated by the ability of 2-azido-ATP to serve as a substrate in the amino acid activation reaction. The modified protein was subjected to tryptic digestion, and the fragments labeled by the nucleotide analogue were purified by reverse-phase high-performance liquid chromatography. Sequence analysis identified three tryptic peptides corresponding to residues G373-K384, W405-R416, and L483-K494, derived from the N-terminal half of the TY1 sequence. As this region shows similarity to strongly conserved regions in other peptide synthetases and acyl-CoA synthetases, it is considered to be the region catalyzing aminoacyl adenylate formation. The identified sequences appear to define components of the nucleotide binding domain found in close proximity to the adenine ring in ATP. Conservation of primary structure and homology to other carboxyl-activating enzymes of this superfamily, including peptide synthetases, insect luciferases, and acyl-CoA synthetases, is discussed.

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