Proteins Encoded bySphingomonas elodeaATCC 31461rmlAandugpGGenes, Involved in Gellan Gum Biosynthesis, Exhibit both dTDP- and UDP-Glucose Pyrophosphorylase Activities

Abstract

The commercial gelling agent gellan is a heteropolysaccharide produced by Sphingomonas elodea ATCC 31461. In this work, we carried out the biochemical characterization of the enzyme encoded by the first gene (rmlA) of the rml 4-gene cluster present in the 18-gene cluster required for gellan biosynthesis (gel cluster). Based on sequence homology, the putative rml operon is presumably involved in the biosynthesis of dTDPrhamnose, the sugar necessary for the incorporation of rhamnose in the gellan repeating unit. Heterologous RmlA was purified as a fused His 6 -RmlA protein from extracts prepared from Escherichia coli IPTG (isopropyl-␤-D-thiogalactopyranoside)-induced cells, and the protein was proven to exhibit dTDP-glucose pyrophosphorylase (K m of 12.0 M for dTDP-glucose) and UDP-glucose pyrophosphorylase (K m of 229.0 M for UDPglucose) activities in vitro. The N-terminal region of RmlA exhibits the motif G-X-G-T-R-X 2 -P-X-T, which is highly conserved among bacterial XDP-sugar pyrophosphorylases. The motif E-E-K-P, with the conserved lysine residue (K 163 ) predicted to be essential for glucose-1-phosphate binding, was observed. The S. elodea ATCC 31461 UgpG protein, encoded by the ugpG gene which maps outside the gel cluster, was previously identified as the UDP-glucose pyrophosphorylase involved in the formation of UDP-glucose, also required for gellan synthesis. In this study, we demonstrate that UgpG also exhibits dTDP-glucose pyrophosphorylase activity in vitro and compare the kinetic parameters of the two proteins for both substrates. DNA sequencing of ugpG gene-adjacent regions and sequence similarity studies suggest that this gene maps with others involved in the formation of sugar nucleotides presumably required for the biosynthesis of another cell polysaccharide(s).Sphingomonas elodea (formerly Pseudomonas elodea and also referred to as Sphingomonas paucimobilis) ATCC 31461 synthesizes the exopolysaccharide (EPS) gellan, a gelling agent with applications in the food, pharmaceutical, and other industries (13,41). This EPS is composed of a repeating linear tetrasaccharide unit consisting of D-glucose (Glc), D-glucuronic acid (GlcA), and L-rhamnose (Rha) in a 2:1:1 ratio, respectively, with glycerate and acetate substituents (22). The significant changes in rheology observed upon the deacylation of gellan are essentially due to the glycerate substituents (17).The gellan repeat unit is formed by sequential transfer of the sugar nucleotides to a membrane-anchored lipid carrier by committed glycosyltransferases, followed by gellan polymerization and export (34). The pathway leading to the formation of the sugar nucleotides UDP-Glc, UDP-GlcA, and dTDP-L-Rha, donors of the monomers for assembling the gellan tetrasaccharidic repeat unit, was elucidated (27, 34). Eighteen genes, organized in the gel cluster (15, 34) and coding for enzymes presumably involved in the synthesis of dTDP-L-Rha, glucosyltransferases, and proteins required for gellan polymerization and export, were identified. The organization and the...

CugP Is a Novel Ubiquitous Non-GalU-Type Bacterial UDP-Glucose Pyrophosphorylase Found in Cyanobacteria

“…Notably, it showed practically no activity toward any other substrates or combinations, including Man-1P, even though Sll1558 and related homologs had provisionally been considered to be GDP-Man PPases, as annotated in CyanoBase. dTTP does not substitute for UTP in reactions with Sll1558, a finding that contrasts with the ability of dTTP to act as a substrate of the GalU-type UDP-Glc PPase from Xanthomonas campestris (8) and Sphingomonas elodea ATCC 31461 (31).…”

“…Because CugP-type UDP-Glc PPase is also included in this superfamily, we discuss structural implications of the motif for substrate specificities based on the known structures. These enzymes bind (d)NTP and sugar-1P sequentially in the substratebinding pocket, which can be divided into a (d)NTP-binding site and a sugar-binding site (18,31). In the former site, three highly conserved residues directly interact with the pyrimidine/purine base of (d)NTP by hydrogen bonds (Fig.…”

Identification of Novel Acetyltransferase Activity on the Thermostable Protein ST0452 from Sulfolobus tokodaii Strain 7

“…Several sugar-1-phosphate nucleotidylyltransferases (sugar-1-P NTases) involved in some biosynthetic reactions, such as glucose-1-phosphate thymidylyltransferase (Glc-1-P TTase), N-acetyl-D-glucosamine-1-phosphate uridyltransferase (GlcNAc-1-P UTase), and mannose-1-phosphate guanylyltransferase, have been identified from many bacteria, including pathogens, as well as from eukaryotes (5,10,11,17,25,27,29). However, only limited information on the archaeal enzyme with sugar-1-P NTase activity has been obtained.…”

“…The nucleotide-sugar molecule is used as a substrate for the construction of the polymer structure of carbohydrates and as a starting material for the biosynthesis of modified-sugar-nucleotide conjugates; for example, TDP-glucose is the starting material for the construction of TDP-rhamnose (25). Several sugar-1-phosphate nucleotidylyltransferases (sugar-1-P NTases) involved in some biosynthetic reactions, such as glucose-1-phosphate thymidylyltransferase (Glc-1-P TTase), N-acetyl-D-glucosamine-1-phosphate uridyltransferase (GlcNAc-1-P UTase), and mannose-1-phosphate guanylyltransferase, have been identified from many bacteria, including pathogens, as well as from eukaryotes (5,10,11,17,25,27,29).…”

The Complex of Sphingomonas elodea ATCC 31461 Glucose-1-Phosphate Uridylyltransferase with Glucose-1-Phosphate Reveals a Novel Quaternary Structure, Unique among Nucleoside Diphosphate-Sugar Pyrophosphorylase Members

“…The bacterium Sphingomonas elodea ATCC 31461 (25,30) is an industrially used microorganism for the high-yield synthesis of exopolysaccharide gellan gum, approved in the United States and European Union as a food gelling, stabilizing, and suspending agent, either on its own or in combination with other hydrocolloids (9). In its native form, gellan is a linear anionic heteropolysaccharide based on a tetrasaccharide repeat unit composed of two molecules of D-glucose (D-Glc), one of D-glucuronic acid (D-GlcA), and one of L-rhamnose (LRha).…”