Structural and biochemical interrogation on transketolase fromPichia stipitisfor new functionality

Abstract

In the development of new functionalities of transketolase for the industrial strain Pichia stipitis (TKps) the structural information of TKps would allow us to gain insight into the enzyme's reaction mechanisms, substrates selectivity and reaction directionality to help reach the goal. We here report seven TKps crystal structures of wild type (WT) and mutants in complex with various physiological ligands. These complexes were refined to resolutions at 1.6-1.03 Å. Both biochemical and mutagenic analyses concluded that residues His27, His66, His100, His261, His478, Asp473, Arg356 and Arg525 play important roles in coenzyme binding and substrates recognition. In general, His66 and His261 hold thiamine diphosphate in place; Arg356 and Arg525 serve as gatekeepers interacting with the terminal phosphate group of sugar-phosphates. His27, His66, His100, His478 and Asp473 are critical for sugars recognition/binding, in which His27 is relatively more important in interaction with sedoheptulose-7-phosphate (S7P) than xylulose-5-phosphate (X5P) in terms of molecular recognition/binding affinity. Kinetically, the reactions with X5P (forward) which were catalyzed by WT or H27A are indistinguishable, while in the reactions with S7P (backward) H27A exhibits weaker activity relative to WT. As a result, given TKps(H27A) as the biocatalyst the overall reactivity reverses from the backward reaction preference to forward, thus facilitating net xylose assimilation.

Phage Display on the Anti‐infective Target 1‐Deoxy‐d‐xylulose‐5‐phosphate Synthase Leads to an Acceptor–Substrate Competitive Peptidic Inhibitor

“…For ThDP‐dependent enzymes such as TK, the binding pockets of ThDP and the acceptor substrate are often in proximity and even share some key amino acids (Figure S3). This might also apply to DXS and its acceptor substrate, d ‐glyceraldehyde‐3‐phosphate ( d ‐GAP).…”

“…For ThDP-dependente nzymes such as TK,t he binding pockets of ThDPa nd the acceptor substrate are often in proximity and even share some key amino acids [36][37][38][39][40][41][42] (Figure S3). This might also apply to DXS and its acceptor substrate, d-glyceraldehyde-3-phosphate (d-GAP).T oi dentify both substrate-and cofactor-competitive inhibitors during screening, the concentrations of both ThDP and d-GAP shouldb ea djusted accordingly upon evaluation of the inhibitory activity.…”

Evidence of Diradicals Involved in the Yeast Transketolase Catalyzed Keto‐Transferring Reactions

“…The bound protein was then eluted with elution buffer (10 mL; 20 m m Tris at pH 8, 500 m m NaCl, 250 m m imidazole). The yield of protein was about 10 mg L −1 , of which the enzymatic activity was examined by using the conventional enzyme‐coupled assay (triosephosphate isomerase and glycerol‐3‐phosphate dehydrogenase) to monitor the production/consumption of G3P . Gel filtration was performed by using an Äkta FPLC system equipped with an S‐200 Superdex column (Amersham Bioscience) under isocratic conditions (20 m m Tris, pH 8, 100 m m NaCl).…”