Structural basis for the activity of two muconate cycloisomerase variants toward substituted muconates

We have refined to 2.3 Angstrom resolution two muconate cycloisomerase (MCI ase) Variant structures, F329I and I54V, that differ from each other and fr om wild-type in their activity toward cis,cis-muconate (CCM) and substitute d CCMs. The working and free R-factors for F329I are 17.4/21.6% and for I54 V, 17.6/22.3% with good stereochemistry. Except for the mutated residue, th ere are no significant changes in structure, To understand the differences in enzymatic properties we docked substituted CCMs and CCM into the active sites of the variants and wild type. The extra space the mutations create a ppears to account for most of the enzymatic differences, The lack of other structural changes explains why, although structurally equivalent changes o ccur in chloromuconate cycloisomerase (CMCIase), the changes in themselves do not convert a MCIase into a dehalogenating CMCIase. Reanalysis of the CM CIase structure revealed only one general acid/base, K169, The structural i mplication is that, in a-chloro-CCM conversion by CMCIase, the lactone ring of 5-chloromuconolactone rotates before dehalogenation to bring the acidic C4 proton next to K169, Therefore, K169 alone performs both required proto nation and deprotonation steps, the first at C5 as in MCIase, and the secon d, after ring rotation, at C4. This distinguishes CMCIase from alpha/beta b arrel isomerases and racemases, which use two different bases. (C) 1999 Wil ey-Liss, Inc.