RESUMEN
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-ß1,4-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.