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Improving alternate lignin catabolite utilization of LigAB from Sphingobium sp. strain SYK-6 through site directed mutagenesis

•Mutation of LigAB enhances use of lignin derived aromatic compounds.•Multiple mutants have enhanced catalytic efficiency with 3OMG.•F103H has enhanced catalytic efficiency for both 3OMG and GA.•Phe103α LigAB mutants outperform native catalyst DesZ for 3OMG dioxygenation. Protocatechuate 4,5-dioxyge...

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Published in:Process biochemistry (1991) 2015-10, Vol.50 (10), p.1634-1639
Main Authors: Barry, Kevin Patrick, Cohn, Erin Francis, Ngu, Abraham, Taylor, Erika Anne
Format: Article
Language:English
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Summary:•Mutation of LigAB enhances use of lignin derived aromatic compounds.•Multiple mutants have enhanced catalytic efficiency with 3OMG.•F103H has enhanced catalytic efficiency for both 3OMG and GA.•Phe103α LigAB mutants outperform native catalyst DesZ for 3OMG dioxygenation. 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 α subunit (Phe103α) controls substrate specificity through interaction with the C5-funtionality of bound substrates, and mutagenesis would enhance GA and 3OMG catalysis. LigAB with Phe103α mutations (F103V, F103T and F103H) displayed enhanced catalytic efficiency for dioxygenation of 3OMG, with mutants displaying 12- to 31-fold increases in kcatapp/Kmapp, 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.
ISSN:1359-5113
1873-3298
DOI:10.1016/j.procbio.2015.05.024