Dehydro valine

Addition of DL-a-aminoadipic acid-2- C to fermentations of the Cephalo-sporium sp. in a complex medium resulted in the production of cephalosporin C with i C localized in the a-aminoadipoyl side-chain (Trown et aL, 1963). Under similar conditions C from dl-valine-1- C was incorporated into the a/8-dehydro-valine fragment of cephalosporin C (Trown et aL, 1963), while acetate-l- C was incorporated, as expected, into the acetoxy group of the molecule and also into the a-aminoadipoyl side-chain (Trown et aL, 1962). The C from L-vahne- C and also from a mixture of dl- and w socystine-3- C was incorporated into cephalosporin C produced by suspensions of washed mycelium with an efficiency similar to that of its incorporation into peniciUin N (Trown et aL, 1963 Abraham et aL, 1964). 

In most cases of formation of peptides containing d-amino acid, the L form of the amino acid is the substrate for the incorporating enzyme. In contrast, the free D-amino acid is ordinarily a poor substrate for the incorporation reaction. Whether the racemization occurs on the enzyme or afterwards remains to be determined in most cases. In the case of the D-valyl residue formed in penicillin, a tripeptide derivative containing L-valine is an intermediate, and the conversion is thought to occur by way of an a,/3-dehydro form of the valyl residue. 

Stable isotope methodology has been applied to the study of the biosynthesis of madumycin II (A2315A, 92) in Actinoplanes philippensis (60, 64). As with virginiamycin Ml (90), carbons 2, 26, 27, and 28 were found to be derived from valine, C-29 from methionine, C-3 to C-6 from acetate, N-7, C-8, and C-9 from glycine, carbons 10 to 17 and C-31 from acetate, and N-18, C-19, C-20, 0-21, C-32, and 0-34 from serine. The origin of the D-alanine residue, N-23, C-24, C-25, C-35, and 0-36, was of particular interest in this study. No incorporation of DL-[U-13C]serine was observed in the alanine portion of the molecule, eliminating the intermediacy of the a,(3-dehydro alanine unit 101 derivable from the acylserine precursor 100. This was corroborated by the observed incorporation into the molecule of intact doubly labeled L-[3-l3C,3,3,3-2H]alanine. dl-[1-l4C]Alanine was also efficiently incorporated. These results and those from de-... 

Peptides in which either component has the D-configuration or is a dehydro amino acid are not attacked. Differences in sensitivity of bonds in protein molecules and in small synthetic substrates indicate that adjacent structures exert great influence on the bonds that may be attacked. For example, one leucine-valine bond in insulin (whose amino acid sequence is known) is hydrolyzed rapidly, whereas this sequence in another part of the insulin molecule and in synthetic substrates resists hydrolysis. ... 

Alternatively, the loss of the a-hydrogen atom of valine during incorporation could be due to a 2,3-dehydro-intermediate. Further evidence for such an intermediate was provided by Adriens et al. (216) who synthesised DL-[3- H]-valine and fed it with DL-[4- C]-valine, to a P. chrysogenum culture. The resulting phenoxymethylpenicillin was purified and combusted to separate the and labels as and HaO. It was found that only about 1% of the tritium label had been incorporated into the penicillin relative to incorporation. The possibility that the loss... 

In summary the D-valinyl moiety of the penicillin molecule is derived from L-valine. The epimerisation during incorporation does not involve deamination. A 2,3-dehydrovalinyl intermediate may be involved but a 3,4-dehydro intermediate is not. The cm-dimethyl groups of valine are incorporated into penicillin with retention of stereochemistry. 

Starting L-[2,3- H]-valine had been retained in the D-valine. Obviously the a-tritium had been lost during epimerisation but as the C-3 tritium had been retained a 2,3-dehydro-intermediate could not be involved. Adriens et al 249) reached a similar conclusion using P. chrysogenum. These authors fed L-[l- C-2,3,4- H]-valine to washed mycelium and then isolated the LLD-tripeptide. By comparing the ratios of the labelled L-valine and the... 

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