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.

Figure 1

Role of sulfoquinovosidases (SQases) in allowing sulfoglycolytic utilization of sulfoquinovose glycosides. (a) Sulfoglycolysis pathways in bacteria highlighting proposed role of sulfoquinovidases. (b) Cartoon of active-site residues involved in binding PNPSQ, from the X-ray structure of E. coli YihQ D472N.

Figure 2

Sulfoquinovosyl glycerol (SQGro) is a superior substrate to sulfoquinovose (SQ) for growth of E. coli. (a) NMR time course of hydrolysis of SQGro hydrolysis by E. coli YihQ. (b) Rates of consumption of individual SQGro diastereoisomers by YihQ. (c) Growth of E. coli BW25113 in M9 minimal media containing 4 mM Glc, Gro, SQGro, or SQ as sole carbon source at 30 °C. (d) MS/MS spectrum of DHPS produced in culture media of E. coli grown on SQGro.

Figure 3

Structures of SQ-derived substrates, ligands, and analogues.

Figure 4

Structural basis of SQ recognition by SQases. (a) Overlay of EcYihQ and AtSQase. (b) Comparison of Michaelis complexes of acid/base mutants of EcYihQ and AtSQase. (c) IFGSQ bound to AtSQase. (d) IFGSQ bound to EcYihQ. For electron density maps see Supporting Information, Figure S5.

Figure 5

(a) Sequence logo highlighting relative proportions of different residues found at each position within the QQRWY/KERWY motif of SQases, using the 84 sequences of Figure 7. (b) Kinetic analysis of mutants investigating the effect of stepwise variation of QQ/KE sequence of EcYihQ and AtSQase. Footnote a: saturation was not reached.

Figure 6

(a) Dendrogram of interrelationships between sequence positions of EcYihQ and AtSQase. Coevolving groups are highlighted in colored boxes. (b) Spatial distribution of three pairs of coevolving residues on the 3D structures. Residues identified by MISTIC based on mutual information are presented in similar colors. Residue 451 exhibits natural variation in NCBI/RefSeq entries WP_010972911.1 (Ile; used for the X-ray structure herein) and WP_035199431.1 (Leu).

Figure 7

Evolutionary relationships for putative SQases. (Right) A phylogenetic tree of putative SQases obtained via multiple sequence alignment presenting a conserved KERWY/QQRWY motif. The alignment of the motif region is depicted together with the positions of the other two coevolving residue pairs identified. Organism taxonomy (class level) is also depicted. Sequences were highlighted by colored boxes based on motif conservation in two main groups: in blue those that presented the KERWY motif and in red those that presented the QQRWY motif. The yellow box groups sequences that in general do not present a conserved arginine, and the remaining sequences from plants and fungi were grouped in green. (Left) A sequence logo of the KERWY/QQRWY motif supplemented with the aforementioned coevolving pairs. Figure generated with WebLogo 3.5.0.