Identification and functional characterization of transketolases in sulfoglycolytic pathways

Transketolase (TK, EC 2.2.1.1) is an essential thiamine pyrophosphate (TPP)-dependent enzyme that plays a central role in carbohydrate metabolism, particularly in the pentose phosphate pathway (PPP) and the photosynthesis Calvin cycle. TK catalyzes a reversible transfer of a two-carbon ketol group (C2 moiety) between phosphorylated sugars, influencing metabolic flux in central carbon metabolism. In addition, TK has evolved specialized roles in sulfoglycolysis-a pathway critical for degrading the plant-derived sulfonated sugar sulfoquinovose (SQ) and sustaining global sulfur cycling. In anaerobes, the sulfoglycolytic transketolase-dependent (sulfo-TK) pathway uses SqwGH (EC 2.2.1.15), a TK encoded by a split-gene sqwG and sqwH, to catalyze two ketol transfers: first from 6-deoxy-6-sulfofructose (SF) to d-glyceraldehyde-3-phosphate (G3P), yielding 4-deoxy-4-sulfoerythrose (SE) which further undergoes aldose-ketose isomerization to generate 4-deoxy-4-sulfoerythrulose (SEu) for the second SqwGH-mediated transketolation. Here, we outline the identification, expression, purification, and activity assay of the split-gene encoded SqwGH. These approaches provide a comprehensive toolkit for researchers to dissect TK's evolutionary plasticity, and engineer its catalytic promiscuity for biocatalytic applications.