Evidence of Kinetic Cooperativity in Dimeric Ketopantoate Reductase from Staphylococcus aureus.

Ketopantoate reductase (KPR) catalyzes the NADPH-dependent production of pantoate, an essential precursor in the biosynthesis of coenzyme A. Previous structural studies have been limited to Escherichia coli KPR, a monomeric enzyme that follows a sequential ordered mechanism. Here we report the crystal structure of the Staphylococcus aureus enzyme at 1.8 Å resolution, the first description of a dimeric KPR. Using sedimentation velocity analysis, we show that the S. aureus KPR dimer is stable in solution. In fact, our structural analysis shows that the dimeric assembly we identify is present in the majority of KPR crystal structures. Steady state analysis of S. aureus KPR reveals strong positive cooperativity with respect to NADPH (Hill coefficient of 2.5). In contrast, high concentrations of the substrate ketopantoate (KP) inhibit the activity of the enzyme. These observations are consistent with a random addition mechanism in which the initial binding of NADPH is the kinetically preferred path. In fact, Förster resonance energy transfer studies of the equilibrium binding of NADPH show only a small degree of cooperativity between subunits (Hill coefficient of 1.3). Thus, the apparently strong cooperativity observed in substrate saturation curves is due to a kinetic process that favors NADPH binding first. This interpretation is consistent with our analysis of the A181L substitution, which increases the Km of ketopantoate 844-fold, without affecting kcat. The crystal structure of KPRA181L shows that the substitution displaces Ser239, which is known to be important for the binding affinity of KP. The decrease in KP affinity would enhance the already kinetically preferred NADPH binding path, making the random mechanism appear to be sequentially ordered and reducing the kinetic cooperativity. Consistent with this interpretation, the NADPH saturation curve for KPRA181L is hyperbolic.

Characterization of Thermotoga maritima glycerol dehydrogenase for the enzymatic production of dihydroxyacetone.

NAD-dependent Thermotoga maritima glycerol dehydrogenase (TmGlyDH) converts glycerol into dihydroxyacetone (DHA), a valuable synthetic precursor and sunless tanning agent. In this work, recombinant TmGlyDH was characterized to determine if it can be used to catalyze DHA production. The pH optima for glycerol oxidation and DHA reduction at 50 °C were 7.9 and 6.0, respectively. Under the conditions tested, TmGlyDH had a linear Arrhenius plot up to 80 °C. TmGlyDH was more thermostable than other glycerol dehydrogenases, remaining over 50 % active after 7 h at 50 °C. TmGlyDH was active on racemic 1,2-propanediol and produced (R)-1,2-propanediol from hydroxyacetone with an enantiomeric excess above 99 %, suggesting that TmGlyDH can also be used for chiral synthesis. (R)-1,2-propanediol production from hydroxyacetone was demonstrated for the first time in a one-enzyme cycling reaction using glycerol as the second substrate. Negative cooperativity was observed with glycerol and DHA, but not with the cofactor. Apparent kinetic parameters for glycerol, DHA, and NAD(H) were determined over a broad pH range. TmGlyDH showed little activity with N(6)-carboxymethyl-NAD(+) (N(6)-CM-NAD), an NAD(+) analog modified for easy immobilization to amino groups, but the double mutation V44A/K157G increased catalytic efficiency with N(6)-CM-NAD(+) ten-fold. Finally, we showed for the first time that a GlyDH is active with immobilized N(6)-CM-NAD(+), suggesting that N(6)-CM-NAD(+) can be immobilized on an electrode to allow TmGlyDH activity in a system that reoxidizes the cofactor electrocatalytically.