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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.

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 (k(cat)) for reductions of bulky-bulky ketones at constant substrate and coenzyme affinities (i.e. K(m Ketone), K(m NADH)).

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., The Saccharomyces cerevisiae gene encoding xylulose kinase (XKS1) was over-expressed to an abundance of ⩾10% intracellular protein in Escherichia coli. Instability of XKS1, not pointed out in previous reports of the enzyme, prevented isolation of active enzyme in native or “tagged” form under a wide range of purification conditions. A fusion protein haboring C-terminal Strep-tag II (XKS1-Strep) displayed activity (∼20 U/mg) as isolated. However, the half-life time of purified XKS1-Strep was only ∼1.5 h at 4 °C and could not be enhanced substantially by an assortment of extrinsic stabilizers (osmolytes, protein, substrates). Peptide mass mapping and N-terminal sequencing showed that the recombinant protein was structurally intact, ruling out proteolytic processing and chemical modifications as possible factors to compromise the stability of the enzyme as isolated. Partial functional complementation of a largely inactive XKS1 preparation by the high-molecular mass fraction (⩾10 kDa) of cell extract prepared from an E. coli BL21 (DE3) expression host suggests a possible role for heterotropic protein-XKS1 interactions in conferring activity/stability to the enzyme. Michaelis–Menten constants of XKS1-Strep were determined: d-xylulose (210 ± 40 μM) and Mg2+-ATP (1.70 ± 0.10 mM).