SUBSTRATE-SPECIFICITY OF AND PRODUCT FORMATION BY MUCONATE CYCLOISOMERASES - AN ANALYSIS OF WILD-TYPE ENZYMES AND ENGINEERED VARIANTS

Muconate cycloisomerases play a crucial role in the bacterial degradat ion of aromatic compounds by converting cis,cis-muconate, the product of catechol ring cleavage, to (4S)-muconolactone, Chloromuconate cyclo isomerases catalyze both the corresponding reaction and a dehalogenati on reaction in the transformation of chloroaromatic compounds, This st udy reports the first thorough examination of the substrate specificit y of the muconate cycloisomerases from Pseudomonas putida PRS2000 and Acinetobacter ''calcoaceticus'' ADP1. We show that they transform, in addition to cis,cis-muconate, 3-fluoro-, 2-methyl-, and 3-methyl-cis,c is-muconate with high specificity constants but not 2-fluoro-, 2-chlor o-, 3-chloro-, or 2,4-dichloro-cis,cis-muconate. Based on known three- dimensional structures, variants of P, putida muconate cycloisomerase were constructed by site-directed mutagenesis to contain amino acids f ound in equivalent positions in chloromuconate cycloisomerases. Some o f the variants had significantly increased specificity constants for 3 -chloro- or 2,4-dichloromuconate (e,g,, A271S and I54V showed 27- and 22-fold increases, respectively, for the former substrate). These kine tic improvements were not accompanied by a change from protoanemonin t o cis,cis-dienelactone as the product of 3-chloro-cis,cis-muconate con version. The rate of 2-chloro-cis,cis-muconate turnover was not signif icantly improved, nor was this compound dehalogenated to any significa nt extent. However, the direction of 2-chloro-cis,cis-muconate cyclois omerization could be influenced by amino acid exchange. While the wild -type enzyme discriminated only slightly between the two possible cycl oisomerization directions, some of the enzyme variants showed a strong preference for either (+)-2-chloro- or (+)-5-chloromuconolactone form ation. These results show that the different catalytic characteristics of muconate and chloromuconate cycloisomerases are due to a number of features that can be changed independently of each other.