Converting NAD-Specific Inositol Dehydrogenase to an Efficient NADP-Selective Catalyst, with a Surprising Twist

By Hudson Roth
Spotlight #2

Zheng, H. et al. Biochemistry 2013, 52, 5876-5883.

a Mutant myo-Inositol dehydrogenase (IDH, EC from Bacillus subtilis binds NADP in a twisted conformation.
a Mutant myo-Inositol dehydrogenase (IDH, EC from Bacillus subtilis binds NADP in a twisted conformation.

In recent years there has been an increasing interest in biocatalysis, the use of enzymes to perform organic transformations, as it is environmentally-friendly and often more selective than traditional organic chemistry methods. Dehydrogenases, which oxidize alcohols in a NAD- or NADP-dependent manner, is one enzyme class commonly used in biocatalysis. Often an additional enzyme is also present in biocatalysis to continually replenish the NAD or NADP needed for the dehydrogenase to function. A specific dehydrogenase is myo-inositol dehydrogenase (IDH) which can be isolated from B. subtilis and is known to oxidize the axial hydroxyl group in myo-inositol using NAD. Previous work by Zheng et al. revealed that IDH retains almost all its activity in 40% DMSO, 15% aqueous MeOH, and after adsorption onto Celite. This prompted them attempt engineering a NADP-dependent IDH mutant as a new coenzyme recycler for biocatalysis.
The only distinction between NAD and NADP is the replacement of the 2’-hydroxyl group on the adenine moiety in NAD by a phosphate in NADP. Therefore the preference of an enzyme for one of these cofactors is due to differences in the adenine-binding portion of the Rossman fold. The crystal structure of NAD-bound IDH revealed Asp35 undergoes a H-bond interaction with the 2’-hydroxyl group of the adenine moiety. The authors replaced this Asp with a Ser via site-directed mutagenesis, resulting in an IDH mutant capable of using both NAD and NADP. The replacement of Asp by a Ser not only eliminated the like-charge repulsion that would occur between the Asp and phosphate, but also provided a potential H-bond interaction between the serine’s hydroxyl group and the phosphate. After examining the Rossman fold of various NADP-dependent dehydrogenases, an additional A12K mutation was introduced into the D35S IDH since most NADP-dependent dehydrogenases contain a positively-charged residue near the orthophosphate portion of NADP. The resulting A12K/D35S IDH mutant exhibited a preference for NADP over NAD and was crystalized bound to NADP. Typically, a dehydrogenase mutated to use NADP over NAD will bind the new cofactor in the same position as the wild-type. However, the crystallography data of the IDH mutant revealed that though the nicotinamide ring remained in the same position, the adenine ring was rotated by 11 angstroms. Zheng et al. argued this unusual binding conformation is likely promoted by a π-stacking interaction between the nitrogenous base and the side-chain of Trp75.
The authors were able to successfully create a NADP-dependent IDH mutant. Not only did Zheng et al. demonstrate how biological molecules can be engineered to alter their biological activity, but also the evolutionary flexibility of the Rossman fold. Mutations in this structural motif of dehydrogenases can potentially confer an advantage to an organism under selective pressure, thus enabling it to survive in a changing environment. Further studies are required to test the ability of this mutant to serve as a new coenzyme recycler in biocatalysis.