Rational design of substrate binding pockets in polyphosphate kinase for use in cost-effective ATP-dependent cascade reactions.

Abstract

Adenosine-5'-triphosphate (ATP) is the energy equivalent of the living system. Polyphosphate (polyP) is the ancient energy storage equivalent of organisms. Polyphosphate kinases (PPKs) catalyze the polyP formation or ATP formation, to store energy or to regenerate ATP, respectively. However, most PPKs are active only in the presence of long polyPs, which are more difficult and more expensive to generate than the short polyPs. We investigated the PPK preference towards polyPs by site-directed mutagenesis and computational simulation, to understand the mechanism and further design enzymes for effective ATP regeneration using short polyPs for in vitro cascade reactions, which are highly desired for research and applications. The results suggest that the short polyPs inhibit PPK by blocking the ADP-binding pocket. Structural comparison between PPK (Corynebacterium glutamicum) and PPK (Sinorhizobium meliloti) indicates that three amino acid residues, i.e., lysine, glutamate, and threonine, are involved in the activity towards short polyP by fixing the adenosine group of ADP in between the subunits of the dimer, while the terminal phosphate group of ADP still offers an active site, which presents a binding pocket for ADP. A proposed triple mutant PPK (SMc02148-KET) demonstrates significant activity towards short polyP to form ATP from ADP. The obtained high glutathione titer (38.79mM) and glucose-6-phosphate titer (87.35mM) in cascade reactions with ATP regeneration using the triple mutant PPK (SMc02148-KET) reveal that the tailored PPK establishes the effective ATP regeneration system for ATP-dependent reactions.