The refined X-ray structure of muconate lactonizing enzyme from Pseudomonas putida PRS2000 at 1.85 Å resolution

Sari Helin, Peter C. Kahn, Bh Lakshmi Guha, Diana G. Mallows, Adrian Goldman

Research output: Contribution to journalArticlepeer-review

55 Scopus citations

Abstract

We report here the refined X-ray crystal structure of muconate lactonizing enzyme (MLE) from Pseudomonas putida PRS2000 at a resolution of 1.85 Å with an R-factor of 16.8%. An enzyme from the β-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 α + β domain, an αβ-barrel domain and a C-terminal meandering subdomain. The αβ-barrel domain is highly irregular. Its structure is (β/α)7β, with the structural role of the last α-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° to -69°; the first and last strands, which close the barrel, cross at an angle of -69°, 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 β-sheet as well as to its neighbouring strands in the barrel. It thus cements the whole protein together. As in other αβ-barrel proteins, the active site of MLE, present in each subunit is at the C-terminal ends of the barrel β-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. Our model thus confirms that Glu327 is the general acid, that Lys169 is the general base, and that the manganese ion and Lys167 provide electrophilic stabilisation of the enol/enolate intermediate that forms during the course of the reaction.

Original languageEnglish (US)
Pages (from-to)918-941
Number of pages24
JournalJournal of molecular biology
Volume254
Issue number5
DOIs
StatePublished - Dec 15 1995

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Structural Biology
  • Molecular Biology

Keywords

  • (α/β)-barrel/β-ketoadipate pathway
  • Muconate lactonizing enzyme
  • Protein structure
  • X-ray crystallography

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