PDBsum entry 1dm3

Transferase

PDB id: 1dm3

Contents

Protein chains

389 a.a. *

Ligands

SO4 ×6

ACO ×4

Waters ×804

* Residue conservation analysis

PDB id:

1dm3

Name:

Transferase

Title:

Acetylated biosynthetic thiolase from zoogloea ramigera in complex with acetyl-coa

Structure:

Biosynthetic thiolase acetylated at cys89. Chain: a, b, c, d. Fragment: entire thiolase, acetylated at cys89. Engineered: yes

Source:

Zoogloea ramigera. Organism_taxid: 350. Cellular_location: cytoplasm. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Tetramer (from PQS)

Resolution:

2.00Å R-factor: 0.214 R-free: 0.256

Authors:

Y.Modis,R.K.Wierenga

Key ref:

Y.Modis and R.K.Wierenga (2000). Crystallographic analysis of the reaction pathway of Zoogloea ramigera biosynthetic thiolase. J Mol Biol, 297, 1171-1182. PubMed id: 10764581 DOI: 10.1006/jmbi.2000.3638

Date:

13-Dec-99 Release date: 29-Aug-01

Protein chains

P07097  (THIL_SHIZO) -  Acetyl-CoA acetyltransferase from Shinella zoogloeoides

Seq: 392 a.a.

Struc: 389 a.a.*

* PDB and UniProt seqs differ at 3 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.2.3.1.9 - acetyl-CoA C-acetyltransferase.
• Pathway: Mevalonate Biosynthesis
• Reaction: 2 acetyl-CoA = acetoacetyl-CoA + CoA

2 × acetyl-CoA (Bound ligand (Het Group name = ACO) corresponds exactly) = acetoacetyl-CoA + CoA

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

reference

DOI no: 10.1006/jmbi.2000.3638

J Mol Biol 297:1171-1182 (2000)

PubMed id: 10764581

Crystallographic analysis of the reaction pathway of Zoogloea ramigera biosynthetic thiolase.

Y.Modis, R.K.Wierenga.

ABSTRACT

Biosynthetic thiolases catalyze the biological Claisen condensation of two acetyl-CoA molecules to form acetoacetyl-CoA. This is one of the fundamental categories of carbon skeletal assembly patterns in biological systems and is the first step in many biosynthetic pathways including those which generate cholesterol, steroid hormones and ketone body energy storage molecules. High resolution crystal structures of the tetrameric biosynthetic thiolase from Zoogloea ramigera were determined (i) in the absence of active site ligands, (ii) in the presence of CoA, and (iii) from protein crystals which were flash frozen after a short soak with acetyl-CoA, the enzyme's substrate in the biosynthetic reaction. In the latter structure, a reaction intermediate was trapped: the enzyme was found to be acetylated at Cys89 and a molecule of acetyl-CoA was bound in the active site pocket. A comparison of the three new structures and the two previously published thiolase structures reveals that small adjustments in the conformation of the acetylated Cys89 side-chain allow CoA and acetyl-CoA to adopt identical modes of binding. The proximity of the acetyl moiety of acetyl-CoA to the sulfur atom of Cys378 supports the hypothesis that Cys378 is important for proton exchange in both steps of the reaction. The thioester oxygen atom of the acetylated enzyme points into an oxyanion hole formed by the nitrogen atoms of Cys89 and Gly380, thus facilitating the condensation reaction. The interaction between the thioester oxygen atom of acetyl-CoA and His348 assists the condensation step of catalysis by stabilizing a negative charge on the thioester oxygen atom. Our structure of acetyl-CoA bound to thiolase also highlights the importance in catalysis of a hydrogen bonding network between Cys89 and Cys378, which includes the thioester oxygen atom of acetyl-CoA, and extends from the catalytic site through the enzyme to the opposite molecular surface. This hydrogen bonding network is different in yeast degradative thiolase, indicating that the catalytic properties of each enzyme may be modulated by differences in their hydrogen bonding networks.

Selected figure(s)

Figure 2.
Figure 2. Stereo picture of the biosynthetic thiolase tetramer with its "tetrahedral cage" tetramerization motif. The tetramer is colored according to the B-factor of the C^a atom of each residue. Dark blue corresponds to a B-factor of vert, similar 5 Å2; red corresponds to 100 Å2. The A-B dimer (upper half of the Figure) has significantly lower B-factors than the C-D dimer (lower half). This is a result of the layered packing in the crystal lattice [Modis and Wierenga 1999]. Four molecules of acetyl-CoA are shown in ball-and-stick representation, bound to each of the four subunits near the interface between the A-B and C-D dimers. The side-chains of residues located in high B-factor loops and pointing towards the CoA moiety are also shown. These include residues Lys133, Arg172, Lys208 and Arg232-Pro233-Ala234-Phe235-Asp236-Lys237, which are all located in the loop domain. Figure 2, Figure 5 and Figure 6 were prepared with MOLSCRIPT [Kraulis 1991] and Raster3D [Merritt and Bacon 1997].

Figure 7.
Figure 7. Schematic drawing of the hydrogen bonding network extending from the acitve site of biosynthetic thiolase, through the enzyme to the molecular surface opposite the active site pocket. Water molecules are shown as gray circles.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2000, 297, 1171-1182) copyright 2000.

Figures were selected by an automated process.