Biochemical characterization and phylogenetic analysis of UDP-glucose dehydrogenase from the gellan gum producer Sphingomonas elodea ATCC 31461

摘要

Sphingomonas elodea ATCC 31461 synthesizes in high yield the exopolysaccharide gellan, which is a water-soluble gelling agent with many applications. In this study, we describe the cloning and sequence analysis of the ugdG gene, encoding a UDP-glucose dehydrogenase (47.2 kDa; UDPG-DH; EC 1.1.1.22), required for the synthesis of the gellan gum precursor UDP-glucuronic acid. UgdG protein shows homology to members of the UDP-glucose/GDP-mannose dehydrogenase superfamily. The Neighbor-Joining method was used to determine phylogenetic relationships among prokaryotic and eukaryotic UDPG-DHs. UgdG from S. elodea and UDPG-DHs from Novosphingobium, Zymomonas, Agrobacterium, and Caulobacter species form a divergent phylogenetic group with a close evolutionary relationship with eukaryotic UDPG-DHs. The ugdG gene was recombinantly expressed in Escherichia coli with and N-terminal 6-His tag and purified for biochemical characterization. The enzyme has an optimum temperature and pH of 37°C and 8.7, respectively. The estimated apparent K m values for UDP-glucose and NAD+ were 0.87 and 0.4 mM, respectively. DNA sequencing of chromosomal regions adjacent to ugdG gene and sequence similarity studies suggests that this gene maps together with others presumably involved in the biosynthesis of S. elodea cell wall polysaccharides.