Polypeptide synthetase

F Schauwecker, F Pfennig, N Grammel, U Keller. Construction and in vitro analysis of a new bi-modular polypeptide synthetase for synthesis of N-methylated acyl peptides. Chem Biol 7 287-297, 2000. 

The heparan, chondroitin and hyaluronan synthetases, although one polypeptide chain, have two active sites corresponding to the two glycosyl transfers catalysed. Kinetic studies are technically very difficult, but the point was established in the case of the hyalurononan and chondroitin synthetases by the construction of chimeric proteins. Pasteurella multocida produces both chondroitin and hyaluronan by the action of two-domain single polypeptide synthetases. The chimeric protein from the N-terminal half of the hyaluronan synthetase and the C-terminal half of the chondroitin synthetase produced only... 

It was earlier considered that all the amino acid-activating synthetases were derived from a single primeval synthetase , so that all synthetases would have similar structures. Surprisingly, however, this is not the case. When the primary sequences, and in part the secondary and tertiary structures, of all the synthetases had been determined, clear differences in their construction became obvious. The aminoacyl-tRNA synthetases consist either of one single polypeptide chain (a) or of two or four identical polypeptides (ot2 or 04). In addition, there are heterogeneously constructed species with two sets of two identical polypeptide chains (OC2P2). This nomenclature indicates that, for each synthetase, a or P refers to a primary structure. The number of amino acids can vary from 334 to more than 1,000. 

Figure 7 The direct and indirect pathways of tRNA asparaginylation. The direct pathway consists of charging by AsnRS on tRNA " of free Asn formed with asparagine synthetase A or B. The Asn-tRNA " binds the EF-Tu factor in bacteria (or EF-1A in eukaryotes and archaea) to be carried to the ribosome, in the indirect pathway, a nondiscriminating AspRS (ND-AspRS) charges Asp on tRNA " Asp-tRNA " does not bind the eiongation factor but is converted by the tRNA-dependent trimeric amidotransferase GatCAB into Asn-tRNA ", which binds the EF-Tu factor and is carried to the ribosome where it is used for polypeptide chain elongation.

Building on earlier work of Osawa and co-workers [55], Oliver and Kowal [52] tested the feasibility of introducing a noncoded amino acid at an unassigned codon in M. luteus. DNA templates were prepared which coded for 19-mer polypeptides containing either the unassigned codon AGA(Arg) or the termination codon TAG at position 13 under the control of a T7 RNA polymerase promoter. The corresponding tRNAs, produced as described in Sect. 2, were based on tRNA and acylated with phenylalanine. The tRNA was modified to prevent recognition by the alanine aminoacyl-tRNA synthetase and to increase translational efficiency. 

Figure 20.24 The physiological pathway of polypeptide synthesis. The flux-generating step is that catalysed by the aminoacyl-tRNA synthetases, indicated by the broad arrow. The assumption implicit in this interpretation is that the physiological pathway starts with the intracellular amino acids and ends with the peptide that is formed in the elongation and termination processes. For the majority of enzymes, the concentration of intracellular amino acids is higher than the K, for the synthetase (Chapter 3).

The translation of the mRNA into proteins is the final step in the biological flow of information (see Fig. 6.1). Similar to other macromolecular polymerizations, protein synthesis can be divided into initiation, chain elongation, and termination. Critical players in this process are the aminoacyl transfer RNAs (tRNAs). These molecules form the interface between the mRNA and the growing polypeptide. Activation of tRNA involves the addition of an amino acid to its acceptor stem, a reaction catalyzed by an aminoacyl-tRNA synthetase. Each aminoacyl-tRNA synthetase is highly specific for one amino acid and its corresponding tRNA molecule. The anticodon loop of each aminoacyl-tRNA interacts... 

Proofreading by Aminoacyl-tRNA Synthetases The amino-acylation of tRNA accomplishes two ends (1) activation of an amino acid for peptide bond formation and (2) attachment of the amino acid to an adaptor tRNA that ensures appropriate placement of the amino acid in a growing polypeptide. The identity of the amino acid attached to a tRNA is not checked on the ribosome, so attachment of the correct amino acid to the tRNA is essential to the fidelity of protein synthesis. 

The transformylase is more selective than the Met-tRNA synthetase it is specific for Met residues attached to tRNAfMet, presumably recognizing some unique structural feature of that tRNA. By contrast, Met-tRNAMet inserts methionine in interior positions in polypeptides. 

Many enzymes are named by adding the suffix -ase to a word, or words, descriptive of the type of enzymatic activity. Thus, esterases hydrolyze esters, proteinoses hydrolyze proteins, reductases achieve reductions, and synthetases achieve syntheses of polypeptide chains, nucleic acid chains, and other molecules. 

The amino acids in the cytoplasm will not form polypeptides unless activated by ester formation with appropriate tRNA molecules. The ester linkages are through the 3 -OH of the terminal adenosine nucleotide (Equation 25-9) and are formed only under the influence of a synthetase enzyme that is specific for the particular amino acid. The energy for ester formation comes from ATP hydrolysis (Sections 15-5F and 20-10). The product is called an amino-acyl-tRNA. 

Bennett, M.J., Lightfoot, D.A. Cullimore, J.V. (1989). cDNA sequence and differential expression of the gene encoding the glutamine synthetase y polypeptide of Phaseolus vulgaris L. Plant Molecular Biology 12, 553-65. 

Lara, M., Porta, H., Padilla, J., Folch, J. Sanchez, F. (1984). Heterogeneity of glutamine synthetase polypeptides in Phaseolus vulgaris L. Plant Physiology 76, 1019-23. 

Tingey, S.V., Walker, E.L. Coruzzi, G.M. (1987). Glutamine synthetase genes of pea encode distinct polypeptides which are differentially expressed in leaves, roots and nodules. The EMBO Journal 6, 1-9. 

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