How genes are expressed and translated into proteins (using mRNA, codons and tRNAs as adaptor molecules) forms the basis of the 'genetic code'. Many peptides are synthesized nonribosomally, however, by large protein complexes that also serve as templates. Recent advances have shed light on what the nonribosomal code is and how it can be read.

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...

SUMMARYWatermelon is a crop with a high water demand and is frequently grown under conditions of higher than normal root-zone salinity. In the present study, seedlings of watermelon (cv. Fantasy, Citrullus lanatus (Thunb.) Matsum & Nakai) were grown either ungrafted or grafted on three rootstocks: Strong Tosa, S1 (both Cucurbita maxima×Cucurbita moschata), or Emphasis (Lagenaria siceraria). All the plants were exposed to an NaCl-induced salinity stress (electrical conductivity, EC=2·2, 4·0, or 6·0 dS/m). The vegetative growth of all the plants substantially reduced after 2 weeks of exposure to 6·0 dS/m; however, growth of the plants grafted on Strong Tosa reduced less than that of the others. The leaf water content and specific leaf area (SLA, m2/g) decreased with an increasing salinity in grafted plants, but not in ungrafted plants. Salinity induced an increase of superoxide dismutase (SOD) activity in grafted plants up to two-fold depending on the rootstock, whereas it had no effect on this enzyme activity in ungrafted plants. Leaf Na+ concentration increased with increasing salinity in ungrafted and S1 grafted plants, whereas there was no significant leaf Na+ accumulation in Emphasis and Strong Tosa grafted plants. Leaf K+ concentration was affected by the rootstock but not by salinity, thus, the ability to keep a high K+/Na+ ratio was achieved mainly by limiting leaf Na+ concentration. The rootstock determined the leaf Cl− accumulation, with lower overall concentrations found if plants were grafted on the S1 rootstock than on Emphasis or ungrafted plants. Salinity significantly decreased the leaf NO3− concentration on Emphasis grafted plants only, while the NO3−/Cl− ratio was reduced in all the rootstocks. The capacity of Strong Tosa to withstand salt stress better than other tested rootstocks was probably due to the ability to induce anatomical adaptation (SLA) and SOD activity in response to salt stress, and also to the efficiency of Na+ exclusion from the shoot.

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