An efficient 2'-O- to 3'-C-β-d-glucosidic bond rearrangement on the dihydrochalcone phloretin to convert phlorizin into nothofagin was achieved by combining complementary O-glycosyltransferase (OGT) and C-glycosyltransferase (CGT) activities in a one-pot transformation containing catalytic amounts of uridine 5'-diphosphate (UDP). Two separate enzymes or a single engineered dual-specific O/CGT were applied. Overall (quantitative) conversion occurred in two steps via intermediary UDP-glucose and phloretin.
Highlights► Xylulose kinase (XKS1) is a key enzyme for xylose utilization in Saccharomyces cerevisiae. ► XKS1 was recombinantly produced in E. coli, and native and tagged forms of the enzyme were isolated. ► XKS1 was highly unstable, losing its activity rapidly during purification or storage. ► Isolated XKS1 was shown by MS to be structurally intact. ► XKS1 harboring a C-terminal Strep-tag II was purified with retention of activity and characterized.
Aldo-keto reductases tighten coenzyme binding by forming a hydrogen bond across the pyrophosphate group of NAD(P)(H). Mutation of the hydrogen bonding anchor Lys24 in Candida tenuis xylose reductase prevents fastening of the “safety belt” around NAD(H). The loosened NAD(H) binding leads to increased turnover numbers (kcat) for reductions of bulky-bulky ketones at constant substrate and coenzyme affinities (i.e. Km Ketone, Km NADH).
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