Combined enzymatic synthesis of nucleotide (deoxy) sugars from sucrose and nucleoside monophosphates

Zervosen, Astrid; Stein, Andréas; Adrian, Holger; Elling, Lothar

doi:10.1016/0040-4020(95)01081-5

Cited by 37 publications

(17 citation statements)

References 23 publications

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“…In those processes, adenylate kinase was used for the conversion of each NMP to NDP. However, adenylate kinase does not show any substrate specificity for dTMP, resulting in the difficulty in the use of dTMP for dTDP-sugar production (Zervosen et al, 1996). We have overcome this problem by employing the recombinant TMP-kinase for the conversion of dTMP to dTDP.…”

Section: Discussionmentioning

confidence: 98%

One‐pot enzymatic production of dTDP‐4‐Keto‐6‐Deoxy‐d‐glucose from dTMP and glucose‐1‐phosphate

Lee

Kim

et al. 2003

Biotech & Bioengineering

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An enzymatic production method for dTDP-4-keto-6-deoxy-D-glucose, a key intermediate of various deoxysugars in antibiotics, was developed starting from dTMP, acetyl phosphate, and glucose-1-phosphate. Four enzymes, i.e., TMP kinase, acetate kinase, dTDP-glucose synthase, and dTDP-D-glucose 4,6-dehydratase' were overexpressed using T7 promoter system in the E. coli BL21 strain, and the dTDP-4-keto-6-deoxy-D-glucose was synthesized by using the enzyme extracts in one-pot batch system. When 20 mM dTMP of initial concentration was used, Mg2+ ion, acetyl phosphate, and glucose-1-phosphate concentrations were optimized. About 95% conversion yield of dTDP-4-keto-6-deoxy-D-glucose was obtained based on initial dTMP concentration at 20 mM dTMP, 1 mM ATP, 60 mM acetyl phosphate, 80 mM glucose-1-phosphate, and 20 mM MgCl(2). The rate-limiting step in this multiple enzyme reaction system was the dTDP-glucose synthase reaction. Using the reaction scheme, about 1 gram of purified dTDP-4-keto-6-deoxy-D-glucose was obtained in an overall yield of 81% after two-step purification, i.e., anion exchange chromatography and gel filtration.

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Section: Discussionmentioning

confidence: 98%

One‐pot enzymatic production of dTDP‐4‐Keto‐6‐Deoxy‐d‐glucose from dTMP and glucose‐1‐phosphate

Lee

Kim

et al. 2003

Biotech & Bioengineering

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“…A further advantage of the SuSy system is the prolonged stability of all employed enzymes in the presence of 300-500 mM sucrose enabling us to use them in a repetitive batch mode with high enzyme productivities [100][101][102][103]. Starting from 41 and dTMP (108), 13 was synthesized in an overall yield of 62% (with reference to dTMP) which corresponds to 430 µmol (253 mg) [230].…”

Section: Chemo-enzymatic Synthesismentioning

confidence: 99%

Nucleotide Deoxysugars: Essential Tools for the Glycosylation Engineering of Novel Bioactive Compounds

Rupprath¹,

Schumacher²,

Elling

2005

CMC

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The irreversible spread of new resistance mechanisms against existing therapeutical antibiotics has led to the development of technologies and strategies for the glycosylation engineering of novel antibiotics. Amino-, C-branched and O-methylated 6-deoxyhexoses play a favourite role in the biosynthesis of clinically important antibiotics like tylosin, erythromycin or oleandomycin and are essential for the antibiotic activity. They are transferred onto the aglycon by glycosyltransferases using dTDP-activated deoxyhexoses. The in vitro biochemical characterization of the biosynthetic enzymes and the glycosyltransferases are, however, hampered due to the poor synthetic access to dTDP-activated deoxysugars and their biosynthetic intermediates. The overcoming of the poor availability of dTDP-activated sugars was the target of several researchers to fulfil their distinct aims with these sugars which were mostly involved in the synthesis of different biological active compounds. Several completely different strategies were used in the past years to improve the availability of dTDP-activated deoxysugars, varying from complete enzymatic synthesis via syntheses using reaction technology for yield optimization to full organic synthesis or shortcuts like the decomposition of commercially available antibiotics and later chemical activation of the sugar moieties. This review gives a survey of the synthesis of dTDP-activated sugars by chemical and chemoenzymatic approaches and discusses the promiscuity of glycosyltransferases to evaluate the chances for applying them for the production of new bioactive compounds. It summarizes the most important enzymes in the field of synthesis using biosynthetic pathway enzymes and describes solutions for occurring challenges during application. Finally, this review will give a survey about the availability of dTDP-activated sugars in sufficient scale and will also point at important sugars which are still bottlenecks and difficult to synthesize and therefore should become a target for enhanced research efforts.

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“…Thus we established the first economic syntheses of different NDP-a-d-glucoses (N = U, dU, C, A) from nucleoside monophosphates (NMP) and sucrose through combinations of monophosphate kinase and myokinase, respectively, and SuSy. [40,49] In this paper, we report on a flexible enzyme module system starting from dTMP and sucrose for the preparative (chemo)enzymatic synthesis of the precursor 2, the intermediate 3, dTDPb-l-rhamnose (4), dTDP-a-d-quinovose (6) and dTDP-a-dfucose (7). The enzyme module system also allows the biochemical characterization of enzymes from deoxysugar pathways.…”

Section: Introductionmentioning

confidence: 99%

An Enzyme Module System for the Synthesis of dTDP‐activated Deoxysugars from dTMP and Sucrose

et al. 2005

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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.

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Combined enzymatic synthesis of nucleotide (deoxy) sugars from sucrose and nucleoside monophosphates

Cited by 37 publications

References 23 publications

One‐pot enzymatic production of dTDP‐4‐Keto‐6‐Deoxy‐d‐glucose from dTMP and glucose‐1‐phosphate

One‐pot enzymatic production of dTDP‐4‐Keto‐6‐Deoxy‐d‐glucose from dTMP and glucose‐1‐phosphate

Nucleotide Deoxysugars: Essential Tools for the Glycosylation Engineering of Novel Bioactive Compounds

An Enzyme Module System for the Synthesis of dTDP‐activated Deoxysugars from dTMP and Sucrose

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