TY - JOUR

T1 - An enzyme module system for the synthesis of dTDP-activated deoxysugars from dTMP and sucrose

AU - Elling, Lothar

AU - Rupprath, Carsten

AU - Günther, Nicole

AU - Römer, Ulrike

AU - Verseck, Stefan

AU - Weingarten, Petra

AU - Dräger, Gerald

AU - Kirschning, Andreas

AU - Piepersberg, Wolfgang

PY - 2005/8/5

Y1 - 2005/8/5

N2 - A flexible enzyme module system is presented that allows preparative access to important dTDP-activated deoxyhexoses from dTMP and sucrose. The strategic combination of the recombinant enzymes dTMP-kinase and sucrose synthase (SuSy), and the enzymes RmlB (4,6-dehydratase), RmlC (3,5-epimerase) and RmlD (4-ketoreductase) from the biosynthetic pathway of dTDP-β-L-rhamnose was optimized. The SuSy module (dTMP-kinase, SuSy, ± RmlB) yielded the precursor dTDP-α-D-glucose (2) or the biosynthetic intermediate dTDP-6-deoxy-4-keto-α-D-glucose (3) on a 0.2-0.6 g scale with overall yields of 62% and 72%, respectively. A two-step strategy in which the SuSy module was followed by the deoxysugar module (RmlC and RmlD) resulted in the synthesis of dTDP-β-L-rhamnose (4; 24.1 μmol, overall yield: 35.9%). Substitution of RmlC by DnmU from the dTDP-β-L-daunosamine pathway of Streptomyces peucetius in this module demonstrated that DnmU acts in vitro as a 3,5-epimerase with 3 as substrate to yield 4 (32.2 μmol, overall yield: 44.7%). Chemical reduction of 3 with NaBH4 gave a mixture of the C-4 epimers dTDP-α-D-quinovose (6) and dTDP-α-D-fucose (7) in a ratio of 2:1. In summary, the modular character of the presented enzyme system provides valuable compounds for the biochemical characterization of deoxysugar pathways playing a major role in microbial producers of antibiotic and antitumour agents.

AB - A flexible enzyme module system is presented that allows preparative access to important dTDP-activated deoxyhexoses from dTMP and sucrose. The strategic combination of the recombinant enzymes dTMP-kinase and sucrose synthase (SuSy), and the enzymes RmlB (4,6-dehydratase), RmlC (3,5-epimerase) and RmlD (4-ketoreductase) from the biosynthetic pathway of dTDP-β-L-rhamnose was optimized. The SuSy module (dTMP-kinase, SuSy, ± RmlB) yielded the precursor dTDP-α-D-glucose (2) or the biosynthetic intermediate dTDP-6-deoxy-4-keto-α-D-glucose (3) on a 0.2-0.6 g scale with overall yields of 62% and 72%, respectively. A two-step strategy in which the SuSy module was followed by the deoxysugar module (RmlC and RmlD) resulted in the synthesis of dTDP-β-L-rhamnose (4; 24.1 μmol, overall yield: 35.9%). Substitution of RmlC by DnmU from the dTDP-β-L-daunosamine pathway of Streptomyces peucetius in this module demonstrated that DnmU acts in vitro as a 3,5-epimerase with 3 as substrate to yield 4 (32.2 μmol, overall yield: 44.7%). Chemical reduction of 3 with NaBH4 gave a mixture of the C-4 epimers dTDP-α-D-quinovose (6) and dTDP-α-D-fucose (7) in a ratio of 2:1. In summary, the modular character of the presented enzyme system provides valuable compounds for the biochemical characterization of deoxysugar pathways playing a major role in microbial producers of antibiotic and antitumour agents.

KW - Antibiotics

KW - Biocatalysis

KW - Carbohydrates

KW - Enzymes

KW - Glycoconjugates

UR - http://www.scopus.com/inward/record.url?scp=23644432034&partnerID=8YFLogxK

U2 - 10.1002/cbic.200500037

DO - 10.1002/cbic.200500037

M3 - Article

C2 - 15977277

AN - SCOPUS:23644432034

VL - 6

SP - 1423

EP - 1430

JO - ChemBioChem

JF - ChemBioChem

SN - 1439-4227

IS - 8

ER -