Structural and Biochemical Insights into the Function and Evolution of Sulfoquinovosidases

Abstract

An estimated 10 billion tonnes of sulfoquinovose (SQ) are produced and degraded each year. Prokaryotic sulfoglycolytic pathways catabolize sulfoquinovose (SQ) liberated from plant sulfolipid, or its delipidated form α-d-sulfoquinovosyl glycerol (SQGro), through the action of a sulfoquinovosidase (SQase), but little is known about the capacity of SQ glycosides to support growth. Structural studies of the first reported SQase (Escherichia coli YihQ) have identified three conserved residues that are essential for substrate recognition, but crossover mutations exploring active-site residues of predicted SQases from other organisms have yielded inactive mutants casting doubt on bioinformatic functional assignment. Here, we show that SQGro can support the growth of E. coli on par with d-glucose, and that the E. coli SQase prefers the naturally occurring diastereomer of SQGro. A predicted, but divergent, SQase from Agrobacterium tumefaciens proved to have highly specific activity toward SQ glycosides, and structural, mutagenic, and bioinformatic analyses revealed the molecular coevolution of catalytically important amino acid pairs directly involved in substrate recognition, as well as structurally important pairs distal to the active site. Understanding the defining features of SQases empowers bioinformatic approaches for mapping sulfur metabolism in diverse microbial communities and sheds light on this poorly understood arm of the biosulfur cycle.

A sulfoglycolytic Entner-Doudoroff pathway inRhizobium leguminosarumbv.trifoliiSRDI565

“…The activity in the YSQ-derived lysate was inhibited by the addition of IFG-SQ, an azasugar inhibitor of SQases that makes key interactions in the active site that mimic those required for substrate recognition (Figure 4c). 24 The similar levels of activity of SQase in both mannitol and SQ grown Rl -SRDI565 is consistent with the abundance of the putative SQase WP_017967311.1 detected by proteomic analysis.…”

“…The chromogenic substrate 4-nitrophenyl α-sulfoquinovoside (PNPSQ), which was designed as an analogue of the natural substrate SQGro, results in release of the chromophore 4-nitrophenolate, which can be detected using UV-visible spectrophotometry at 400 nm. 23, 24 Rl -SRDI565 was grown to mid-logarithmic phase in Y 5% M and Y 5% SQ media, and the harvested cells used to prepare a cell-free lysate containing soluble proteins. Incubation of Y 5% M and Y 5% SQ-derived lysates with PNPSQ both resulted in production of 4-nitrophenolate at similar rates.…”

A Sulfoglycolytic Entner-Doudoroff Pathway in Rhizobium leguminosarum bv. trifolii SRDI565

“…Direct evidence for enzymatic activity associated with the sulfo-ED operon in Rl-SRDI565 was obtained by measuring SQase enzyme activity in cell lysates. The chromogenic substrate 4-nitrophenyl -sulfoquinovoside (PNPSQ), which was designed as an analogue of the natural substrate SQGro, results in release of the chromophore 4-nitrophenolate, which can be detected using UV-visible spectrophotometry with high sensitivity at 400 nm or at the isosbestic point, 348 nm (24,25). Rl-SRDI565 was grown to mid-logarithmic phase in Y 5% M and Y 5% SQ media, and the harvested cells used to prepare a cell-free lysate containing soluble proteins.…”

Environmental and Intestinal Phylum Firmicutes Bacteria Metabolize the Plant Sugar Sulfoquinovose via a 6-Deoxy-6-sulfofructose Transaldolase Pathway

“…We suggest that for the SFT pathway, the glycerol acquired from SQG cleavage may directly feed into generation of the acceptor molecule GAP for the SF transaldolase, through glycerol kinase and glycerol-3-phosphate dehydrogenase, some of which are co-encoded in the predicted gene clusters (see Figure 5 ). This needs to be addressed in future, as well as the directly related questions whether indeed SQG rather than SQ might be the more environmentally relevant substrate for SQ-catabolic microorganisms ( Abayakoon et al., 2018 ) and whether the SQG glucosidase gene may be a universal genetic marker for SQ(G) metabolism across all types of pathways.…”