A Chimeric Variant of Two Gentisate 1,2‐Dioxygenases with Improved Catalytic Activity, Altered Structural Conformation and Substrate Specificity

Abstract

Gentisate 1,2-dioxygenases catalyze the critical ring-opening step in the metabolism of gentisate (2,5-dihydroxybenzoate) by microorganisms under aerobic conditions. Gentisate 1,2-dioxgenases from bacterial species that have been characterized to date are strictly Fe(II)-dependent and they are mostly homotetrameric in solution. In bacteria, genes coding for gentisate 1,2-dioxygenase and other enzymes in the rest of the pathway for gentisate degradation are often organized in operons. While genes and enzymes in the gentisate pathway have been relatively well-studied in bacteria, reports about their fungal counterparts remain scarce in the literature. Here, we report the biochemical characterization of two gentisate 1,2-dioxygenases from Aspergillus niger with different specific activities that appear to be encoded within two split gene clusters in the A. niger genome. These differences in activities seem to be accompanied by differences in secondary structure and oligomerization states between these two proteins in solution. Furthermore, we report the engineering of a chimeric variant consisting of two domains derived from these two A. niger gentisate 1,2-dioxgenases that shows improved activity with gentisate, altered structural conformation and substrate specificity able to cleave a substrate that neither of the two wild type gentisate 1,2-dioxygenases is able to attack.