UDP-sulfoquinovose formation by Sulfolobus acidocaldarius

Abstract

The UDP-sulfoquinovose synthase Agl3 from Sulfolobus acidocaldarius converts UDP-d-glucose and sulfite to UDP-sulfoquinovose, the activated form of sulfoquinovose required for its incorporation into glycoconjugates. Based on the amino acid sequence, Agl3 belongs to the short-chain dehydrogenase/reductase enzyme super-family, together with SQD1 from Arabidopsis thaliana, the only UDP-sulfoquinovose synthase with known crystal structure. By comparison of sequence and structure of Agl3 and SQD1, putative catalytic amino acids of Agl3 were selected for mutational analysis. The obtained data suggest for Agl3 a modified dehydratase reaction mechanism. We propose that in vitro biosynthesis of UDP-sulfoquinovose occurs through an NAD+-dependent oxidation/dehydration/enolization/sulfite addition process. In the absence of a sulfur donor, UDP-d-glucose is converted via UDP-4-keto-d-glucose to UDP-d-glucose-5,6-ene, the structure of which was determined by 1H and 13C-NMR spectroscopy. During the redox reaction the cofactor remains tightly bound to Agl3 and participates in the reaction in a concentration-dependent manner. For the first time, the rapid initial electron transfer between UDP-d-glucose and NAD+ could be monitored in a UDP-sulfoquinovose synthase. Deuterium labeling confirmed that dehydration of UDP-d-glucose occurs only from the enol form of UDP-4-keto-glucose. The obtained functional data are compared with those from other UDP-sulfoquinovose synthases. A divergent evolution of Agl3 from S. acidocaldarius is suggested.

Functional Characterization of Enzymatic Steps Involved in Pyruvylation of Bacterial Secondary Cell Wall Polymer Fragments

“…Briefly, purified P. alvei MnaA-His 6 (45 μg) was incubated with UDP-GlcNAc (Sigma-Aldrich) at a concentration of 1.3 or 0.5 mM, in 46 mM sodium phosphate buffer, pH 7.5, supplemented with 11 mM MgCl 2 , in a total reaction volume of 192.5 μl at 37°C for up to 30 min; individual reactions were done and stopped in intervals of 5 min. After heat-inactivation (100°C, 1 min), the mixture was centrifuged (10,000 × g , 5 min, RT), and the supernatant was analyzed by reversed-phase (RP) HPLC (Thermo Scientific/Dionex; Ultimate 3000 Standard LC System) on a Hyperclone 5 μ ODS column (Phenomenex, 150 mm × 4.6 mm, 5 μ) using 0.4 M sodium phosphate buffer, pH 6.1, with a flow rate of 0.6 ml/min as eluent ( Zolghadr et al, 2015 ). Peaks were identified using UDP (1 nmol), UDP-GlcNAc (5 nmol), and UDP-ManNAc (5 nmol) as standards; detection was done at 254 nm.…”

Entner–Doudoroff pathway for sulfoquinovose degradation in Pseudomonas putida SQ1

“…Although the biosynthesis of SQ and SQDG in plants, algae, cyanobacteria, and archaea has been studied in considerable detail (e.g., 23,24), much less is known about the degradation reactions and the recycling of the carbon and sulfur bound in SQ and SQDG. The lipid can easily be hydrolyzed by plant acyl-hydrolases, which liberate SQ-glycerol (1), and glucosidases can liberate SQ in the next step (25); however, it is still unclear whether higher plants are capable of splitting the carbon/sulfur bond in SQ at significant rates.…”

Nucleotide sugar dehydratases: Structure, mechanism, substrate specificity, and application potential

“…TunA from Streptomyces lysosuperificus was the first reported UDP-GlcNAc 5,6-dehydratase (104,105). The 5,6-dehydratase activity was later also observed in 2 more enzymes, namely in Pen from Bacillus thuringiensis, also a UDP-GlcNAc 5,6-dehydratase (122), and in the UDPsulfoquinovose synthase in the absence of sulfite (converts UDP-Glc) (123).…”

“…The UDP-sulfoquinovose synthase (Agl3 (131) or SQD1 ( 124)) normally converts UDP-Glc and sulfite to UDP-sulfoquinovose (Figure 3, purple). This activated form of sulfoquinovose is required for its incorporation into glycoconjugates or glycoprotein N-glycans (123,124,131). Recent structure determination and point mutation studies of Agl3 allowed in-depth mechanistic analyses (123).…”