An enzyme module system for the synthesis of dTDP-activated deoxysugars from dTMP and sucrose.
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-beta-L-rhamnose was optimized. The SuSy module (dTMP-kinase, SuSy, +/-RmlB) yielded the precursor dTDP-alpha-D-glucose (2) or the biosynthetic intermediate dTDP-6-deoxy-4-keto-alpha-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-beta-L-rhamnose (4; 24.1 micromol, overall yield: 35.9 %). Substitution of RmlC by DnmU from the dTDP-beta-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 mumol, overall yield: 44.7 %). Chemical reduction of 3 with NaBH4 gave a mixture of the C-4 epimers dTDP-alpha-D-quinovose (6) and dTDP-alpha-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.
Elling
Lothar
L
Department of Biotechnology/Biomaterial Sciences and Helmholtz Institute for Biomedical Engineering, RWTH Aachen, Worringerweg 1, 52056 Aachen, Germany. l.elling@biotec.rwth-aachen.de
Rupprath
Carsten
C
Günther
Nicole
N
Römer
Ulrike
U
Verseck
Stefan
S
Weingarten
Petra
P
Dräger
Gerald
G
Kirschning
Andreas
A
Piepersberg
Wolfgang
W
D001483:Base Sequence; D002237:Carbohydrate Dehydrogenases / Q000737:chemistry / Q000378:metabolism; D002238:Carbohydrate Epimerases / Q000737:chemistry / Q000378:metabolism; D003837:Deoxy Sugars / Q000096:biosynthesis*; D004792:Enzyme Precursors / Q000737:chemistry* / Q000378:metabolism*; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics; D006836:Hydro-Lyases / Q000737:chemistry / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009702:Nucleoside Diphosphate Sugars / Q000737:chemistry / Q000378:metabolism; D009703:Nucleoside-Phosphate Kinase / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013395:Sucrose / Q000737:chemistry*; D013938:Thymidine Monophosphate / Q000737:chemistry* / Q000378:metabolism; D013942:Thymine Nucleotides / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins
D002237:Carbohydrate Dehydrogenases; D002238:Carbohydrate Epimerases; D003837:Deoxy Sugars; D004792:Enzyme Precursors; D005964:Glucosyltransferases; D006836:Hydro-Lyases; D009702:Nucleoside Diphosphate Sugars; D009703:Nucleoside-Phosphate Kinase; D011994:Recombinant Proteins; D013395:Sucrose; D013938:Thymidine Monophosphate; D013942:Thymine Nucleotides; C073293:thymidine diphosphate rhamnose; C107144:dTDP-4-dehydrorhamnose reductase; C022699:sucrose synthase; C020093:dTMP kinase; C114534:4a-hydroxy-erythro-5,6,7,8-tetrahydrobiopterin dehydratase; C020092:dTDP-4-ketorhamnose 3,5-epimerase; D001426:Bacterial Proteins