Arashdeep Kaur, R. Williams, Shuxin Yang, Mihwa Lee, Niccolay Madiedo Soler, Ethan D. Goddard‐Borger, G.J. Davies, Mahima Sharma, Spencer J. Williams
ABSTRACT Sulfoquinovose (SQ) is a major biogenic sulfonated sugar whose degradation fuels microbial sulfur and carbon cycling. In the sulfoglycolytic Entner–Doudoroff (sulfo‐ED) pathway, 2‐keto‐3,6‐dideoxy‐6‐sulfogluconate (KDSG) is cleaved by KDSG aldolase to yield pyruvate and sulfolactaldehyde, yet the structure and mechanism of this enzyme have remained unclear. We report the biochemical and structural characterization of a metal‐dependent KDSG aldolase from Pseudomonas putida using chemo‐enzymatically synthesized KDSG. The enzyme forms a homohexamer, with a (β/α) 8 TIM‐barrel monomer assembling as a ‘dimer‐of‐trimers’. The enzyme exhibits optimal activity in the presence of Co 2+ or Mn 2+ , consistent with other class II aldolases. Kinetic analysis revealed millimolar‐range K M values for KDSG and modest cross‐reactivity with the related glycolytic intermediate, 2‐keto‐3,6‐deoxy‐6‐phosphogluconate (KDPG). Crystal structures of the apo and Co 2+ •pyruvate‐bound forms (2.85 Å and 2.80 Å) show a metal‐coordinated active site at the subunit interface, with conserved residues mediating metal binding and catalysis, providing insights into the mechanism of sulfonate‐specific aldol cleavage. Sequence‐similarity network and genome‐context analyses show that KDSG aldolases are widespread among Proteobacteria and typically cluster with sulfo‐ED pathway genes. These results define the structural and mechanistic basis of KDSG aldolases and inform on their roles in bacterial sulfur metabolism.