DTDP dihydrostreptose

As Streptomyces contain high proportions of proteases, a protease inhibitor, diphenylcarbamoyl chloride, was added to the buffer during purification of the dTDP-dihydrostreptose synthase. A partially purified, enzyme preparation from S. griseus could be obtained by removal of nucleic acids with streptomycin and fractionation with ammonium sulfate.40 However, when this enzyme preparation was subjected to gel filtration on a column of Sephadex G-100, enzyme activity was completely lost. By combining certain fractions of the column eluate, enzyme activity could be partially restored. 

A compound having the properties of the dTDP derivative of dihydrostreptose was apparently very labile, because it decomposed readily to a compound tentatively identified as dihydrostreptosyl phosphate. The latter was also very labile, and gave free dihydrostreptose upon attempted paper chromatography. 

It was, therefore, assumed that separation into two, or more, active protein-fractions had occurred on the Sephadex column. As the biosynthesis of dTDP-L-rhamnose and dTDP-L-dihydrostreptose are related,37,39 and as, moreover, a dTDP-L-lyxo-4-hexulose 3,5-epimerase is necessary for the formation of dTDP-L-rhamnose,1718 the Sephadex G-100 fractions were assayed for the presence of the 3,5-epimerase by... 

Scheme 10.—Biosynthesis of dTDP-L-dihydrostreptose from dTDP-D-glucose.

With the possibility of preparing dTDP-L-[U-14C]dihydrostreptose40 (see Section II,4),613,62 the enzymic transfer of the dihydrostreptose moiety to streptidine 6-phosphate could now be tested experimentally.63... 

Streptidine phosphate was incubated with dTDP-L-[U-14C]dihydro-streptose, obtained in situ from dTDP-D-[U-14C]glucose with a cell-free extract from S. griseus. Two new, positively charged, radioactive products were obtained which, upon hydrolysis, gave dihydrostreptose as the only radioactive product. Comparison with a synthetic sample of 0-a-L-dihydrostreptose-(l- 4)-streptidine (43, see Scheme 14) proved that one of the products was identical with this pseudodisaccharide, and the second product was identified as the corresponding 6-phosphate (44). It was concluded that the phospho-rylated product had been partially hydrolyzed during the incubation by streptomycin 6-phosphate phosphatase present in the cell-free extract. No transfer-products were formed in controls in which (a) denatured extract was used,(fe) streptidine phosphate was absent, or(c) streptidine was substituted for streptidine phosphate. 

Scheme 14.—Formation of 0-a-L-Dihydrostreptose-(l— 4)-streptidine 6-Phosphate (44) from dTDP-L-dihydrostreptose and Streptidine 6-Phosphate with a Cell-free Extract from S. griseus.

Further modifications of dihydrostreptose to streptose or S -hydroxystreptose seem to occur on the final condensation products only (cf. Section V.E), rather chan during synthesis of the dTDP-activated precursor. The 5 -hydroxy-group in antibiotic produced by S. glaucescem is unlikely to stem from the original D-glucose, since then the three enzymes StrE, StrM, and StrL would need to have acquired new substrates and even a new reaction mechanism in the case of the dTDP-o-glucose 4,6-dehydratase. 

The synthesis of dTDP-L-dihydrostreptose begins with the formation of dTDP-4-keto-6-deoxyglucose (Fig. 35, see also Fig. 31). 

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