Retuning rieske-type oxygenases to expand substrate range

Abstract

Rieske-type oxygenases are promising biocatalysts for the destruction of persistent pollutants or for the synthesis of fine chemicals. In this work, we explored pathways through which Rieske-type oxygenases evolve to expand their substrate range. BphAE p4, a variant biphenyl dioxygenase generated from Burkholderia xenovorans LB400 BphAE LB400 by the double substitution T335A/F336M, and BphAE RR41, obtained by changing Asn 338, Ile 341, and Leu 409 of BphAE p4 to Gln 338, Val 341, and Phe 409, metabolize dibenzofuran two and three times faster than BphAE LB400, respectively. Steady-state kinetic measurements of single- and multiple-substitution mutants of BphAE LB400 showed that the single T335A and the double N338Q/L409F substitutions contribute significantly to enhanced catalytic activity toward dibenzofuran. Analysis of crystal structures showed that the T335A substitution relieves constraints on a segment lining the catalytic cavity, allowing a significant displacement in response to dibenzofuran binding. The combined N338Q/L409F substitutions alter substrate-induced conformational changes of protein groups involved in subunit assembly and in the chemical steps of the reaction. This suggests a responsive induced fit mechanism that retunes the alignment of protein atoms involved in the chemical steps of the reaction. These enzymes can thus expand their substrate range through mutations that alter the constraints or plasticity of the catalytic cavity to accommodate new substrates or that alter the induced fit mechanism required to achieve proper alignment of reactioncritical atoms or groups. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.