PDBsum entry 2bz3

Transferase

PDB id: 2bz3

Contents

Protein chains

406 a.a.

Ligands

NH4 ×4

DAO ×4

SO4 ×2

Waters ×1022

References listed in PDB file

Key reference

Title

Fatty acid synthesis.

Authors

P.Von wettstein-Knowles, J.G.Olsen, K.A.Mcguire, A.Henriksen.

Ref.

FEBS J, 2006, 273, 695-710. [DOI no: 10.1111/j.1742-4658.2005.05101.x]

PubMed id

16441657

Abstract

beta-Ketoacyl-acyl carrier protein (ACP) synthase enzymes join short carbon units to construct fatty acyl chains by a three-step Claisen condensation reaction. The reaction starts with a trans thioesterification of the acyl primer substrate from ACP to the enzyme. Subsequently, the donor substrate malonyl-ACP is decarboxylated to form a carbanion intermediate, which in the third step attacks C1 of the primer substrate giving rise to an elongated acyl chain. A subgroup of beta-ketoacyl-ACP synthases, including mitochondrial beta-ketoacyl-ACP synthase, bacterial plus plastid beta-ketoacyl-ACP synthases I and II, and a domain of human fatty acid synthase, have a Cys-His-His triad and also a completely conserved Lys in the active site. To examine the role of these residues in catalysis, H298Q, H298E and six K328 mutants of Escherichia colibeta-ketoacyl-ACP synthase I were constructed and their ability to carry out the trans thioesterification, decarboxylation and/or condensation steps of the reaction was ascertained. The crystal structures of wild-type and eight mutant enzymes with and/or without bound substrate were determined. The H298E enzyme shows residual decarboxylase activity in the pH range 6-8, whereas the H298Q enzyme appears to be completely decarboxylation deficient, showing that H298 serves as a catalytic base in the decarboxylation step. Lys328 has a dual role in catalysis: its charge influences acyl transfer to the active site Cys, and the steric restraint imposed on H333 is of critical importance for decarboxylation activity. This restraint makes H333 an obligate hydrogen bond donor at N(epsilon), directed only towards the active site and malonyl-ACP binding area in the fatty acid complex.

Figure 1.
Fig. 1. Superimposition of the KAS I C163S-C12 (white, light colors) [3] and KAS I–C8 (gray, dark colors) active sites. Red spheres are water molecules. Blue atoms represent nitrogen, red represent oxygen, and green represents sulfur. Figures 1, 2 and 4 are made in MOLSCRIPT[41].

Figure 3.
Fig. 3. The active sites of the wild-type KAS I, its H298 mutants and their acyl complexes. (A) Wild-type. (B) WT–C8. (C) Superimposition of the wild-type (white, light colors) and H298E (orange, dark colors). (D) H298E. (E) H298E–C12. (F) H298Q. (G) H298Q–C12. (H) Superimposition of H298Q and H298Q–C12. In (A, B) and (D–G), water molecules (red spheres) within hydrogen bonding distance are indicated with dashed lines. (H) Superimposition of H298Q (orange, dark colors) and H298Q–C12 (white, light colors) not including water molecules. Figure prepared using PYMOL[42].

The above figures are reprinted by permission from the Federation of European Biochemical Societies: FEBS J (2006, 273, 695-710) copyright 2006.

Secondary reference #1

Title

Structures of beta-Ketoacyl-Acyl carrier protein synthase i complexed with fatty acids elucidate its catalytic machinery.

Authors

J.G.Olsen, A.Kadziola, P.Von wettstein-Knowles, M.Siggaard-Andersen, S.Larsen.

Ref.

Structure, 2001, 9, 233-243. [DOI no: 10.1016/S0969-2126(01)00583-4]

PubMed id

11286890

Figure 1.
Figure 1. The Three-Step Mechanism Characterizing the Decarboxylating Claisen Condensing EnzymesThe first step is a trans-thioesterification of the primer substrate. Subsequently, malonyl-ACP gets decarboxylated to give the carbanion, which then attacks C1 of the primer substrate, followed by release of the product, 3-oxoacyl-ACP

The above figure is reproduced from the cited reference with permission from Cell Press

Secondary reference #2

Title

Beta-Ketoacyl-[Acyl carrier protein] synthase i of escherichia coli: aspects of the condensation mechanism revealed by analyses of mutations in the active site pocket.

Authors

K.A.Mcguire, M.Siggaard-Andersen, M.G.Bangera, J.G.Olsen, P.Von wettstein-Knowles.

Ref.

Biochemistry, 2001, 40, 9836-9845. [DOI no: 10.1021/bi0105577]

PubMed id

11502177

Secondary reference #3

Title

The X-Ray crystal structure of beta-Ketoacyl [acyl carrier protein] synthase i.

Authors

J.G.Olsen, A.Kadziola, P.Von wettstein-Knowles, M.Siggaard-Andersen, Y.Lindquist, S.Larsen.

Ref.

FEBS Lett, 1999, 460, 46-52. [DOI no: 10.1016/S0014-5793(99)01303-4]

PubMed id

10571059

Figure 3.
Fig. 3. Ribbon structure of the KAS I dimer as viewed perpendicular to the two-fold axis. Helices and strands are red and blue respectively in the subunit on the right and pink and light blue in the subunit on the left. Active site residues Cys-163, His-298, and His-333 are shown in ball and stick representation. The site on the right is accessible from above the page, the other from below. Notice the well-defined separation of secondary structural elements from the capping domain on top and the thiolase core domain below. Constructed using Molscript [42].

Figure 5.
Fig. 5. Superposition of the active sites and presumed binding pockets of KAS I (dark gray sticks) and KAS II (white sticks) in ball-and-stick representation. KAS I residues are labelled with capital letters and KAS II residues in lower case, those marked with ’ are from the other subunit in the dimer. The Cα of KAS II Ile-108 overlaps with the Cα of KAS I Gly-107, and its side chain points into the cavity in KAS I. The KAS II structural equivalent to KAS I Met-197 is Gly-198. The KAS II equivalent to Gln-113’ is Ile-114’ and the space occupied by Glu-200 is partially filled by Phe-133’. Glu-200 and Gln-113’ are at the bottom of the KAS I cavity. The only polar residue in the KAS II binding pocket is Thr-137’ which is an alanine in KAS I.

The above figures are reproduced from the cited reference with permission from the Federation of European Biochemical Societies