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We report here the refined X-ray crystal structure of muconate lactonizing
enzyme (MLE) from Pseudomonas putida PRS2000 at a resolution of 1.85 A with an
R-factor of 16.8%. An enzyme from the beta-ketoadipate pathway, MLE catalyses
the conversion of cis,cis-muconate to muconolactone. It is a homo-octamer, one
monomer consisting of 373 amino acid residues. MLE has two large domains and a
C-terminal subdomain: an alpha + beta domain, an alpha beta-barrel domain and a
C-terminal meandering subdomain. The alpha beta-barrel domain is highly
irregular. Its structure is (beta/alpha)7 beta, with the structural role of the
last alpha-helix being replaced by both the C-terminal subdomain and part of the
N-terminal domain. The fifth, seventh and eighth barrel strands are unusual
because they have left-handed twist about their axes. The strand crossing angles
also vary enormously, from +9 degrees to -69 degrees; the first and last
strands, which close the barrel, cross at an angle of -69 degrees, making
extensive strand-strand hydrogen bonding impossible. The first barrel strand is
also unusual because it starts in the N-terminal domain and forms hydrogen bonds
to the C-terminal subdomain beta-sheet as well as to its neighbouring strands in
the barrel. It thus cements the whole protein together. As in other alpha
beta-barrel proteins, the active site of MLE, present in each subunit is at the
C-terminal ends of the barrel beta-strands. The active site cleft contains an
essential manganese ion, is lined with charged and other polar residues, and
contains many of the crystallographic water molecules. The manganese ion is
octahedrally co-ordinated to three side-chain carboxylate groups and three water
molecules, and is at the centre of a radiating web of ionic and hydrogen-bonding
interactions. Additionally, two water molecules are buried in the centre of the
barrel and two hydrophilic side-chains (Lys167 and Arg196) make both hydrophobic
and hydrophilic packing interactions with much of the barrel interior. The
barrel interior is thus also unusual because it is so hydrophilic; the
dominating force appears to be the need to solvate the metal ion effectively.
This might account for the irregularity of the barrel. The catalytic mechanism
has been investigated by docking both substrate and product in the active site
with the C-COO- of muconolactone superimposed on the corresponding atoms of
cis,cis-muconate. In agreement with earlier kinetic and spectroscopic results,
the manganese ion does not interact directly with substrate or product.(ABSTRACT
TRUNCATED AT 400 WORDS)
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