Abstract:The current methods for regeneration of NAD + and NADP + in dehydrogenase-catalyzed organic synthesis are not technologically mature due to problems with operational stability, kinetic efficiency or thermodynamic equilibrium. We show here that Candida tenuis xylose reductase converts 9,10-phen-A C H T U N G T R E N N U N G anthrenequinone with good turnover frequency when using NADH (12 s À1 ) or NADPH (1.6 s
À1) as the coenzyme and exhibits high binding affinity for this quinone substrate (K m 13 mM). Because chemical reaction of the hydroquinone product with dissolved molecular oxygen regenerates the quinone at a rate comparable to that of the enzymatic reduction, a chemo-enzymatic process is established where the reductase, a catalytic concentration of 9,10-phenanthrenequinone (25 mM) and molecular oxygen promote efficiently the production of NAD(P)+ from NAD(P)H. Oxidation of the hydroquinone occurs via a radical chain reaction that involves superoxide as the propagating species and yields a molar equivalent of hydrogen peroxide for each 9,10-phenanthrenequinone recycled. Using the NAD + -or NADP + -dependent transformation of d-mannitol (25 mM) into d-fructose as a model transformation, the total turnover numbers for 9,10-phenanthrenequinone and the coenzyme obtained in a single batchwise conversion were 1000 and 125, respectively. The yields of ketose product were quantitative, indicating that molecular oxygen reduction drives the thermodynamically unfavourable synthetic reaction. Oxygen transfer to the liquid phase was shown to be rate-limiting for the overall process under conditions of surface aeration and bubble-free molecular oxygen supply. Xylose reductase was fully stable during the reaction (25 8C, pH 8.0). The novel chemo-enzymatic system should therefore be broadly applicable to biocatalytic synthesis with isolated dehydrogenases utilizing NAD + or NADP + .
Substitution of active-site Tyr-51 by Ala (Y51A) disrupted the activity of Candida tenuis xylose reductase by six orders of magnitude. External bromide brought about unidirectional rate enhancement (%2 · 10 3 -fold at 300 mM) for NAD + -dependent xylitol oxidation by Y51A. Activity of the wild-type reductase was dependent on a single ionizable protein group exhibiting a pK of 9.2 ± 0.1 and 7.3 ± 0.3 in the holoenzyme bound with NADH and NAD + , respectively. This group which had to be protonated for xylose reduction and unprotonated for xylitol oxidation was eliminated in Y51A, consistent with a catalytic acid-base function of Tyr-51. Bromide may complement the xylitol dehydrogenase activity of Y51A by partly restoring the original hydrogen bond between the reactive alcohol and the phenolate of Tyr-51.
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.
Despite their widely varying physiological functions in carbonyl metabolism, AKR2B5 (Candida tenuis xylose reductase) and many related enzymes of the aldo-keto reductase protein superfamily utilise PQ (9,10-phenanthrenequinone) as a common in vitro substrate for NAD(P)H-dependent reduction. The catalytic roles of the conserved active-site residues (Tyr51, Lys80 and His113) of AKR2B5 in the conversion of the reactive alpha-dicarbonyl moiety of PQ are not well understood. Using wild-type and mutated (Tyr51, Lys80 and His113 individually replaced by alanine) forms of AKR2B5, we have conducted steady-state and transient kinetic studies of the effects of varied pH and deuterium isotopic substitutions in coenzyme and solvent on the enzymatic rates of PQ reduction. Each mutation caused a 10(3)-10(4)-fold decrease in the rate constant for hydride transfer from NADH to PQ, whose value in the wild-type enzyme was determined as approximately 8 x 10(2) s(-1). The data presented support an enzymic mechanism in which a catalytic proton bridge from the protonated side chain of Lys80 (pK=8.6+/-0.1) to the carbonyl group adjacent to the hydride acceptor carbonyl facilitates the chemical reaction step. His113 contributes to positioning of the PQ substrate for catalysis. Contrasting its role as catalytic general acid for conversion of the physiological substrate xylose, Tyr51 controls release of the hydroquinone product. The proposed chemistry of AKR2B5 action involves delivery of both hydrogens required for reduction of the alpha-dicarbonyl substrate to the carbonyl group undergoing (stereoselective) transformation. Hydride transfer from NADH probably precedes the transfer of a proton from Tyr51 whose pK of 7.3+/-0.3 in the NAD+-bound enzyme appears suitable for protonation of a hydroquinone anion (pK=8.8). These results show that the mechanism of AKR2B5 is unusually plastic in the exploitation of the active-site residues, for the catalytic assistance provided to carbonyl group reduction in alpha-dicarbonyls differs from that utilized in the conversion of xylose.
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