Protocatechuate 4,5-dioxygenase (LigAB) catalyzes dioxygenation of multiple lignin derived aromatic compounds-such as protocatechuate (PCA), gallate (GA) and 3-O-methyl gallate (3OMG)-with decreasing proficiency as the molecule size increases. We predicted that phenylalanine-103 of the ␣ subunit (Phe103␣) controls substrate specificity through interaction with the C5-funtionality of bound substrates, and mutagenesis would enhance GA and 3OMG catalysis. LigAB with Phe103␣ mutations (F103V, F103T and F103H) displayed enhanced catalytic efficiency for dioxygenation of 3OMG, with mutants displaying 12-to 31-fold increases in k app cat /K app m , making these mutant enzymes more active with 3OMG than its native dioxygenase (DesZ). The F103T and F103V point mutants also exhibited allosteric activation for the dioxygenation of PCA and GA, respectively, in the presence of vanillin, as previously observed for LigAB. The enhanced utilization of substrates by these mutants makes them potentially useful for efforts to develop engineered organisms that catabolize lignin into biofuels or fine chemicals.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.