Dynamic Structural Changes Accompany the Production of Dihydroxypropanesulfonate by Sulfolactaldehyde Reductase

Abstract

2,3-Dihydroxypropanesulfonate (DHPS) is a major sulfur species in the biosphere. One important route for the production of DHPS is sulfoglycolytic catabolism of sulfoquinovose (SQ) through the Embden-Meyerhof-Parnas (sulfo-EMP) pathway. SQ is a sulfonated carbohydrate present in plant and cyanobacterial sulfolipids (sulfoquinovosyl diacylglyceride and its metabolites) and is biosynthesised globally at a rate of around 10 billion tonnes per annum. The final step in the bacterial sulfo-EMP pathway involves reduction of sulfolactaldehyde (SLA) to DHPS, catalysed by an NADH-dependent SLA reductase. Based on conserved sequence motifs, we assign SLA reductase to the β-hydroxyacid dehydrogenase (β-HAD) family, an example of a β-HAD enzyme that acts on a sulfonic acid substrate, rather than a carboxylic acid. We report crystal structures of the SLA reductase YihU from E. coli K-12 in its apo and cofactor-bound states, as well as a ternary complex YihU•NADH•DHPS with the cofactor and product bound in the active site. Conformational flexibility observed in these structures, combined with kinetic studies, confirm a sequential mechanism and provide evidence for dynamic domain movements that occur during catalysis. The ternary complex structure reveals a conserved sulfonate pocket in SLA reductase that recognises the sulfonate oxygens through hydrogen bonding to Asn174, Ser178, and the backbone amide of Arg123, along with an ordered water molecule. This triad of residues distinguishes these enzymes from classical β-HADs that act on carboxylate substrates. A comparison of YihU crystal structures with close structural homologues within the β-HAD family highlights key differences in the overall domain organization and identifies a peptide sequence that is predictive of SLA reductase activity.

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

“…Interestingly, the gene clusters of the strictly anaerobic Clostridia encoded, instead of an SLA dehydrogenase (SftD; dark green in Figures 5 and 1 A), exclusively an NADH-dependent DHPS-forming SLA reductase (SftR, indicated in light green in Figures 5 and 1 A), such as the characterized SLA reductases of E. coli (YihU; ref. Denger et al., 2014 ; Sharma et al., 2020 ) and of Desulfovibrio sp. DF1 (DhpA, physiologically catalyzing the reverse reaction; see ref.…”

Molecular Basis of Sulfosugar Selectivity in Sulfoglycolysis

“…While the Ec SQase binding pocket is comprised of Arg301-Trp304-Tyr508(H 2 O), 13 the SLA reductase binding pocket lacks a direct interaction with an Arg; rather, it is comprised of Asn174-Ser178 and the backbone amide of Arg123. 15 …”

“… 7 It was initially described in Escherichia coli and involves the importation of SQ and its glycosides (which undergo intracellular hydrolysis to liberate SQ), catabolic enzymes to produce dihydroxyacetone phosphate (DHAP) and sulfolactaldehyde (SLA), a reductase to convert SLA to dihydroxypropanesulfonate (DHPS), and a permease to export DHPS from the cell. 7 The pathway is encoded by a 10-gene cluster ( ompL and yihO-W , where yihW was renamed csqR ) that encodes a transcriptional regulator (CsqR, formerly YihW); 11 a sulfolipid porin (OmpL) and two transmembrane permeases (YihO, YihP); a sulfoquinovosidase for cleavage of SQ glycosides (YihQ); 12 , 13 an SQ mutarotase (YihR); 14 three core sulfoglycolytic enzymes: SQ-sulfofructose (SF) isomerase (YihS), SF kinase (YihV), and SF-1-phosphate (SFP) aldolase (YihT); and finally SLA reductase (YihU), 15 which forms DHPS for export ( Figure 1 b). The three core sulfoglycolytic enzymes YihS, YihV, and YihT perform roles analogous to three enzymes involved in upper glycolysis: glucose-6-phosphate (G6P) isomerase, phosphofructose kinase (PFK), and fructose bisphosphate (FBP) aldolase, respectively.…”

A microbial sulfoquinovose monooxygenase pathway that enables sulfosugar assimilation

“…The structural studies highlight key residues in the sulfosugar processing enzymes that have evolved to bind this distinguishing group: an Arg283-Tryp286-H2O(Tyr491) triad for recognition of the sulfonate in the SQase; a Thr220-Gly166-Ser43-H2O(His13-Gln46) cluster for recognition of the sulfonate in the SQGro binding protein; and a proposed binding pocket of Trp206-Arg236-His238-Tyr341-His343 for recognition of sulfonate in the SQ monooxygenase. Well-defined sulfonate binding pockets have been highlighted for the enzymes of the sulfo-EMP pathway 8,21,22 and are useful sequence signatures for bioinformatic studies and assignment of the pathways in unstudied organisms.…”