Enzymatic synthesis of TDP-deoxysugars

Abstract

Many biologically active bacterial natural products contain highly modified deoxysugar residues that are often critical for the activity of the parent compounds. Most of these deoxysugars are secondary metabolites that are biosynthesized in the form of nucleotide diphosphate (NDP) sugars prior to their transfer to natural product aglycones by glycosyltransferases. Over the past decade, many biosynthetic pathways that lead to the formation of these unusual sugars have been unraveled, and the mechanisms of many key enzymatic transformations involved in these pathways have been elucidated. However, obtaining workable quantities of NDP-deoxysugars for in vitro studies is often a difficult task. This limitation has hindered an in-depth investigation of the substrate specificity of deoxysugar biosynthetic enzymes, many of which are promiscuous with respect to their NDP-sugar substrates and are, thus, potentially useful catalysts for natural product glycoengineering. Presented in this review are procedures for the enzymatic synthesis and purification of a variety of NDP-deoxysugars, including some early intermediates in NDP-deoxysugar biosynthetic pathways, and highly modified NDP-deoxysugars that are late intermediates in their respective biosynthetic pathways. The procedures described herein could be used as general guidelines for the development of specific protocols for the synthesis of other NDP-deoxysugars.

Scheme 1. Activation of the Glycolytic Intermediate D-Glucose

Sugar activation. D-Glucose (2) and glucose-6-phosphate (3) can be converted to α-D-glucose-1-phosphate (1) by anomeric kinase and phosphohexose mutase, respectively. A nucleotide monophosphate (NMP) moiety is then tranferred from the corresponding NTP to 1 by a nucleotidylyltransferase to from NDP- α-D-glucose (4). Most deoxysugars produced in bacterial secondary metabolism are derived from TDP- α-D-glucose (5).

Scheme 2. Enzymatic Synthesis of TDP- agr;-D-glucose (5)

In the first step of TDP- α-D-glucose (5) synthesis, thymidine triphosphate (TTP) is synthesized from thymidine by three successive ATP-dependent phosphorylations catalyzed by thymidine kinase (TK), thymidylate kinase (TMK), and nucleotide diphosphate kinase (NDK). ATP is only needed in catalytic amounts, due to a pyruvate kinase (PK) ATP regeneration system, which transfers a phosphate group from phosphoenol pyruvate (PEP) to ADP, yielding pyruvate (Pyr) and ATP. Removal of the enzymes and addition of α-D-glucose-1-phosphate (1) and RfbA (a thymidylyltransferase from Salmonella enterica) leads to the synthesis of TDP- α-D-glucose (5).

Scheme 3. Early TDP-deoxysugar Biosynthetic Intermediates

The key intermediate in TDP-deoxysugar biosynthesis is TDP-4-keto-6-deoxy- α-D-glucose (6), which is synthesized from 5 by a TDP-glucose-4,6-dehydratase enzyme (4,6-DH). This intermediate is a branching point for the biosynthetic pathways of TDP-deoxysugars. For 2,6-dideoxyhexoses, 6 is converted to the unstable intermediate 7 by a 2-dehydratse (2-DH). This intermediate can then be reduced by a 3-ketoreductase (3-KR) to give 8 or 9, or it can be transaminated by a 3-aminotransferase (3-AT) to give 10.

Scheme 4. In vitro enzymatic synthesis of highly-modified TDP-deoxysugars

Biosynthetic pathways for several highly modified TDP-deoxysugars (11–14) that have been synthesized in vitro using native pathway enzymes. See text for details.

Scheme 5. Manipulating deoxysugar structures by metabolic pathway engineering

A combination of gene knockout, heterologous gene expression, and precursor feeding experiments allowed the production of several novel compounds in Streptomyces venezuelae. See text for details.