A precise swaying map for how promiscuous cellobiose-2-epimerase operate bi-reaction

Promiscuous enzymes play a crucial role in organism survival and new reaction mining. However, comprehensive mapping of the catalytic and regulatory mechanisms hasn't been well studied due to the characteristic complexity. The cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus (CsCE) with complex epimerization and isomerization was chosen to comprehensively investigate the promiscuous mechanisms. Here, the catalytic frame of ring-opening, cis-enediol mediated catalysis and ring-closing was firstly determined. To map the full view of promiscuous CE, the structure of CsCE complex with the isomerized product glucopyranosyl-β1,4-fructose was determined. Combined with computational calculation, the promiscuity was proved a precise cooperation of the double subsites, loop rearrangement, and intermediate swaying. The flexible loop was like a gear, whose structural reshaping regulates the sway of the intermediates between the two subsites of H377-H188 and H377-H247, and thus regulates the catalytic directions. The different protonated states of cis-enediol intermediate catalyzed by H188 were the key point for the catalysis. The promiscuous enzyme tends to utilize all elements at hand to carry out the promiscuous functions. To comprehensively map the full catalytic view of promiscuous enzyme, the promiscuous CE that exhibits the most complicated and puzzling catalysis was picked for detailed studies. The promiscuous CE was like an intelligent robot with precise cooperation of multiple subsites, flexible loop and protonated intermediates. The flexible loop acts as a gear, swaying the intermediates between the two subsites of H377-H188 (left) and H377-H247 (right) by structural rearrangement. As a result, the catalytic preference between isomerization and epimerization is regulated. Moreover, the different protonated states of the cis-enediol intermediate catalyzed by the H188 were also significant prerequisite for the bifunctions. [Display omitted]