PDBsum entry 1n0h

Lyase

PDB id: 1n0h

Contents

Protein chains

599 a.a. *

Ligands

CIE ×2

DTT ×2

AYD

FAD ×2

TPP

Metals

__K ×2

_MG ×2

Waters ×832

* Residue conservation analysis

PDB id:

1n0h

Name:

Lyase

Title:

Crystal structure of yeast acetohydroxyacid synthase in complex with a sulfonylurea herbicide, chlorimuron ethyl

Structure:

Acetolactate synthase. Chain: a, b. Fragment: mature catalytic subunit. Synonym: acetohydroxy-acid synthase, als, ahas. Engineered: yes

Source:

Saccharomyces cerevisiae. Baker's yeast. Organism_taxid: 4932. Gene: ilv2. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.

Biol. unit:

Dimer (from PQS)

Resolution:

2.80Å R-factor: 0.163 R-free: 0.205

Authors:

S.S.Pang,L.W.Guddat,R.G.Duggleby

Key ref:

S.S.Pang et al. (2004). The crystal structures of Klebsiella pneumoniae acetolactate synthase with enzyme-bound cofactor and with an unusual intermediate. J Biol Chem, 279, 2242-2253. PubMed id: 14557277 DOI: 10.1074/jbc.M304038200

Date:

14-Oct-02 Release date: 07-Jan-03

Protein chains

P07342  (ILVB_YEAST) -  Acetolactate synthase catalytic subunit, mitochondrial from Saccharomyces cerevisiae (strain ATCC 204508 / S288c)

Seq: 687 a.a.

Struc: 599 a.a.

Enzyme reactions

• Enzyme class: E.C.2.2.1.6 - acetolactate synthase.
• Pathway: Isoleucine and Valine Biosynthesis
• Reaction: 2 pyruvate + H⁺ = (2S)-2-acetolactate + CO2

2 × pyruvate + H(+) (Bound ligand (Het Group name = DTT) matches with 55.56% similarity) = (2S)-2-acetolactate + CO2

• Cofactor: Thiamine diphosphate

Thiamine diphosphate (Bound ligand (Het Group name = TPP) corresponds exactly)

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1074/jbc.M304038200

J Biol Chem 279:2242-2253 (2004)

PubMed id: 14557277

The crystal structures of Klebsiella pneumoniae acetolactate synthase with enzyme-bound cofactor and with an unusual intermediate.

S.S.Pang, R.G.Duggleby, R.L.Schowen, L.W.Guddat.

ABSTRACT

Acetohydroxyacid synthase (AHAS) and acetolactate synthase (ALS) are thiamine diphosphate (ThDP)-dependent enzymes that catalyze the decarboxylation of pyruvate to give a cofactor-bound hydroxyethyl group, which is transferred to a second molecule of pyruvate to give 2-acetolactate. AHAS is found in plants, fungi, and bacteria, is involved in the biosynthesis of the branched-chain amino acids, and contains non-catalytic FAD. ALS is found only in some bacteria, is a catabolic enzyme required for the butanediol fermentation, and does not contain FAD. Here we report the 2.3-A crystal structure of Klebsiella pneumoniae ALS. The overall structure is similar to AHAS except for a groove that accommodates FAD in AHAS, which is filled with amino acid side chains in ALS. The ThDP cofactor has an unusual conformation that is unprecedented among the 26 known three-dimensional structures of nine ThDP-dependent enzymes, including AHAS. This conformation suggests a novel mechanism for ALS. A second structure, at 2.0 A, is described in which the enzyme is trapped halfway through the catalytic cycle so that it contains the hydroxyethyl intermediate bound to ThDP. The cofactor has a tricyclic structure that has not been observed previously in any ThDP-dependent enzyme, although similar structures are well known for free thiamine. This structure is consistent with our proposed mechanism and probably results from an intramolecular proton transfer within a tricyclic carbanion that is the true reaction intermediate. Modeling of the second molecule of pyruvate into the active site of the enzyme with the bound intermediate is consistent with the stereochemistry and specificity of ALS.

Selected figure(s)

Figure 2.
FIG. 2. Structure of K. pneumoniae ALS. A shows a ribbon diagram of the overall structure of the resting enzyme tetramer, with the monomers colored green (monomer A), red (B), blue (C), and yellow (D). There is a vertical 2-fold axis of symmetry in this view. The asymmetric unit contains monomers A and B, whereas the active sites are at the AC and BD interfaces. Monomer A is shown in B, with cylinder representations of -helices (red) and -strands (turquoise) shown as arrows, connected by random coil (green). C and D compare the -domains of ALS (C) and AHAS (D), with secondary structure indicated as in B. The residues in contact with FAD (stick model, D) in AHAS and their structural equivalents in ALS (C) are shown in surface representation.

Figure 7.
FIG. 7. The active sites of ALS and AHAS. A shows the active site region of K. pneumoniae ALS (resting enzyme), and B shows the active site of yeast AHAS viewed in a similar orientation. Residues with and without the prime symbol are derived from different monomers. C, the active site of ALS is shown with the second molecule of pyruvate modeled in so that it makes favorable contacts and is orientated so that it would yield the S-enantiomer of acetolactate (D). In this model, the intermediate is represented as the tricyclic carbanion (IVb, Fig. 5), and an alternate conformation of Lys36 is shown for which the -amino group forms an ionic interaction with the carboxylate of the second pyruvate.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2004, 279, 2242-2253) copyright 2004.

Figures were selected by the author.