Novel dTDP-l-Rhamnose Synthetic Enzymes (RmlABCD) From Saccharothrix syringae CGMCC 4.1716 for One-Pot Four-Enzyme Synthesis of dTDP-l-Rhamnose.

Shida Yang,Min Xiao,Xukai Jiang,Li Xu,Zhenxin Yan,Guofeng Gu,Xiaonan An

doi:10.3389/fmicb.2021.772839

Abstract

Deoxythymidine diphospho-l-rhamnose (dTDP-l-rhamnose) is used by prokaryotic rhamnosyltransferases as the glycosyl donor for the synthesis of rhamnose-containing polysaccharides and compounds that have potential in pharmaceutical development, so its efficient synthesis has attracted much attention. In this study, we successfully cloned four putative dTDP-l-rhamnose synthesis genes Ss-rmlABCD from Saccharothrix syringae CGMCC 4.1716 and expressed them in Escherichia coli. The recombinant enzymes, Ss-RmlA (glucose-1-phosphate thymidylyltransferase), Ss-RmlB (dTDP-d-glucose 4,6-dehydratase), Ss-RmlC (dTDP-4-keto-6-deoxy-glucose 3,5-epimerase), and Ss-RmlD (dTDP-4-keto-rhamnose reductase), were confirmed to catalyze the sequential formation of dTDP-l-rhamnose from deoxythymidine triphosphate (dTTP) and glucose-1-phosphate (Glc-1-P). Ss-RmlA showed maximal enzyme activity at 37°C and pH 9.0 with 2.5mMMg2+, and the Km and kcat values for dTTP and Glc-1-P were 49.56μM and 5.39s−1, and 117.30μM and 3.46s−1, respectively. Ss-RmlA was promiscuous in the substrate choice and it could use three nucleoside triphosphates (dTTP, dUTP, and UTP) and three sugar-1-Ps (Glc-1-P, GlcNH2-1-P, and GlcN3-1-P) to form nine sugar nucleotides (dTDP-GlcNH2, dTDP-GlcN3, UDP-Glc, UDP-GlcNH2, UDP-GlcN3, dUDP-Glc, dUDP-GlcNH2, and dUDP-GlcN3). Ss-RmlB showed maximal enzyme activity at 50°C and pH 7.5 with 0.02mM NAD+, and the Km and kcat values for dTDP-glucose were 98.60μM and 11.2s−1, respectively. A one-pot four-enzyme reaction system was developed by simultaneously mixing all of the substrates, reagents, and four enzymes Ss-RmlABCD in one pot for the synthesis of dTDP-l-rhamnose and dUDP-l-rhamnose with the maximal yield of 65% and 46%, respectively, under the optimal conditions. dUDP-l-rhamnose was a novel nucleotide-activated rhamnose reported for the first time.

Highlights

L-rhamnose is a common 6-deoxy hexose found in the cell wall polysaccharides of many bacteria and the carbohydrate moieties of many natural products from bacteria and plants (Halimah et al, 2015; Khalil et al, 2015; Wittgens et al, 2018; Edgar et al, 2019; Garcia-Vello et al, 2020)
Four genes Ss-rmlABCD in the genome of S. syringae China General Microbiological Culture Collection (CGMCC) 4.1716 are annotated to encode the putative dTDP-l-rhamnose synthetic pathway according to the GenBank database
563.0645 562.0825 588.0708 565.0432 564.0587 590.0494 549.0509 548.0671 574.0573 of the glycosyl donor dTDP-l-rhamnose which is difficult to achieve through chemical synthesis

Summary

Introduction

L-rhamnose is a common 6-deoxy hexose found in the cell wall polysaccharides of many bacteria and the carbohydrate moieties of many natural products from bacteria and plants (Halimah et al, 2015; Khalil et al, 2015; Wittgens et al, 2018; Edgar et al, 2019; Garcia-Vello et al, 2020) These rhamnosecontaining biomolecules show great potential in the development of drugs and vaccines owing to their distinct bioactive properties (Wang et al, 2011; Lee et al, 2014; Mei et al, 2019; Pequegnat and Monteiro, 2019). RmlD (E.C.1.1.1.133) is a dTDP-4-keto-rhamnose reductase that reduces dTDP-4keto-rhamnose to dTDP-l-rhamnose in the presence of NADPH

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