The origin of the sugar-F420 dehydrogenase enzyme function: experimental characterization of a new subfamily and ancestral sequence reconstruction

MASCOTTI ML; KUMAR H; LAPADULA WJ; NGUYEN, QT; JURI AYUB M; FRAAIJE, MW

Simposio; VIII Encuentro Regional de Biocatálisis y Biotransformaciones; 2018

F420 is at the same time aversatile and exceptional cofactor in Nature. Its low redox potential makes itperfect to be involved either in reductions or oxidations. Recently, variouskinds of oxidoreductases have been described employing this deazaflavincoenzyme in combination, or not, with other cofactors1. Theluciferase-like subfamily includes the F420 glucose-6-phosphatedehydrogenases (FGDs) and alcohol dehydrogenases (ADHs). They oxidize glucose-6-Por secondary alcohols respectively, with the concomitant production of reducedF420. Besides representing key enzymes in Bacteria and Archaeametabolism, these enzymes are powerful technological tools. In this work, we investigated the evolutionaryhistory of the dehydrogenase subfamily and demonstrated it diversified intothree types of enzymes: the already known FGDs and ADHs, and a third group formedby uncharacterized sequences. By experimentally characterizing two enzymes ofthis group ?substrates profile, steady-state kinetics, optimal conditions foractivity, thermostability- we discovered a broader substrate acceptance. Therefore,this group was named as ?sugar-6-phosphate dehydrogenases? (FSDs). In thiscontext, aiming to understand how the functionality evolved among this whole subfamilyof enzymes, we tackle the reconstruction and experimental characterization ofthe ancestral sequences at the branching points in the phylogeny. By thisapproach, we were able to resurrect the common ancestor between the FGDs andFSDs. This enzyme proved to be more generalist thus less specific. However,most interesting feature was the thermostability 10°C higher than modernenzymes. Our study provides a strong basis for futurework on the discovery of novel F420 dehydrogenases and theengineering of available ones for biotechnological purposes, such as switchingsubstrate specificities or enhancing thermal stabilities2.