Phenylalanine tyrocidine

A second enzyme (of mass 100 kDa) is needed for activation of phenylalanine. It is apparently the activated phenylalanine (which at some point in the process is isomerized from l- to D-phenylalanine) that initiates polymer formation in a manner analogous to that of fatty acid elongation (Fig. 17-12). Initiation occurs when the amino group of the activated phenylalanine (on the second enzyme) attacks the acyl group of the aminoacyl thioester by which the activated proline is held. Next, the freed imino group of proline attacks the activated valine, etc., to form the pentapeptide. Then two pentapeptides are joined and cyclized to give the antibiotic. The sequence is absolutely specific, and it is remarkable that this relatively small enzyme system is able to carry out each step in the proper sequence. Many other peptide antibiotics, such as the bacitracins, tyrocidines,215 and enniatins, are synthesized in a similar way,213 216 217 as are depsipeptides and the immunosuppresant cyclosporin. A virtually identical pattern is observed for formation of polyketides,218 219 whose chemistry is considered in Chapter 21. 

To demonstrate peptide bond formation from aminoacyl adenylates, Dieck-mann [71] has applied the isolated adenylate domain of tyrocidine synthetase 1 to generate various dipeptides. These were obtained from phenylalanyl adenylate with alanine, leucine, leucineamide, and phenylalanine [reaction (16), with A A = amino acid or amine acceptor],... 

Often, cells of a single microbial strain can synthesize more than one member of a chemical family. The final yields of the various members can be shifted by appropriate precursor pressure. The absence or presence of certain growth factors may accomplish this. In the absence of either exogenous phenylalanine or tryptophan, the ratio of tyrocidines A B C synthesized by Bacillus brevis is 1 3 7. If either L- or D-phenylalanine is provided, the main component formed is tyrocidine A. If L- or D-tryptophan is furnished, component D predominates when both phenylalanine and tryptophan are supplied, each of the four components is synthesized. I ... 

Mach, Reich and Tatum were able to demonstrate an inhibition of the biosynthesis of protein in cells of B. brevis by chloramphenicol and puromycin without affecting the synthesis of tyrocidine . Several analogues of amino acids were found, which inhibited the biosynthesis of tyrocidine without affecting that of protein and vice versa. In contrast to protein synthesis, the production of tyrocidine did not depend on the continuous synthesis of RNA. Furthermore, environmental factors were able to control the relative amounts of the different tyrocidines synthesized by genetically homogeneous cultures . Addition of phenylalanine to the culture medium resulted in the almost exclusive synthesis of tyrocidine A, whereas the unsupplemented culture produced tyrocidine A, B and C. In the presence of tryptophan, a new form of tyrocidine, called tyrocidine D, containing three tryptophan in place of three phenylalanine residues, was produced. This lack of absolute requirement for specific amino acids in the formation of a peptide bond is in contrast to the strict specificity of sequential incorporation of amino acids... 

Tyrocidine B, C H N O,]. Purification and amino acid sequence determination King, Craig, J. Am. Chem. Soc. 77, 6624, 6627 (1955). Synthesis Kuromizu, Izumiya, Experi-entia 26, 587 (1970). Possesses the same structure as tyrocidine A except that L-tryptophan replaces the l phenylalanine. Crystals from methanol + isopropyl ether. 

Tyrocidine C, C H N O,. Separation from the tyrocidine mixture Ruttenberg et al. Biochemistry 4, 11 (1965). Possesses same structure as tyrocidine B except that D-tryp -tophan replaces T>-phenylalanine attached to I.-asparagine. Synthesis Kuromizu, Izumiya. Tetrahedron Letters 1970,... 

Under the action of phenylalanine ammonia lyase (EC 4.3.1.5) Phe is transformed to rrans- cinnamic acid. This plays an important role in plants as a precursor of numerous phenolic secondary products such as flav-onoids, lignin, etc. D-Phe is a component of grami-cidin S and tyrocidines. L-Phe tastes weakly bitter, D-Phe tastes sweet. 

Dubos showed that tyrothricin could be separated into two components, gramicidin and tyrocidine. Craig showed that tyrocidine could be resolved into three components, tyrocidines A, B and C. Tyrocidine A has the structure XI and t5nrocidine B differs from XI only in containing an L-tryptophan residue in place of an L-phenylalanine residue . 

The development of cell free systems has played an important role in attempts to decipher the mechanism of peptide antibiotic synthesis ". Most workers agree that this type of synthesis occurs in the complete absence of polynucleotides and that the amino acid sequence is determined by enzyme specificity and organisation in a multi-enzyme complex . The tyrocidines are produced in Bacillus brevis by an enzyme system which has been resolved into three complimentary fractions by Sephadex G-200 filtration a light and an intermediate component (molecular weight 100000 and 230000) which activate L-phenylalanine and L-proline respectively and a heavy fraction (460 000) which activates the remaining eight amino acids including L-phenylalanine. Each... 

Present data indicates that chain growth in both gramicidin S and the tyrocidines commences at the D-phenylalanine residue adjacent to proline and that the first step in the synthesis is the conversion of L-phenylalanine to D-phenyldanine. In the case of the tyrocidines, the synthesis then proceeds in order from the amino to the carboxyl terminus to form a linear decapeptide ending with a thiol ester linked leucine. The peptide then cyclises relatively slowly to the final product. Yamada and Kurahashi showed that the epimerisation of L-phenylalanine in the initial step did not require pyridoxal phosphate or FAD and they suggested that the reaction occurs via the thiol ester enzyme bound form (101), Figure 3.18. 

In an asparagine-glycerol-salts medium, the ratio of tyrocidine A B C is 1 3 7 if L-phenylalanine is added, the main component produced is tyrocidine A (Mach and Tatum, 1964). If L-tryptophane is added to the basal medium, component D is predominant when both L-phenylalanine and L-tryptophane are used, all four components are synthesized. Similar results are obtained with D-phenylalanine and D-tryptophane. 

Furthermore, by addition of appropriate amino acid analogs, it is possible to suppress formation of the peptide antibiotics with very httle alteration of protein synthesis. For example, D-phenylalanine inhibits production of bacitracin (Snoke and Cornell, 1964) threo-j8-phenylserine, j8-2-thienylserine, a-methyl-tryptophan, 5-methyltryptophane, and -fluorophenylalanine suppress tyrocidine synthesis (Mach et aL, I963) and a combination of norvaline, norleucine, and hydroxyproline depresses formation of gramicidin S (Winnick et al., I96I). 

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