Fatty acid synthesis

Abstract

The formation of carbon-carbon bonds is a fundamental biochemical reaction. A number of enzymes involved in various biosynthetic pathways accomplish this by different means. Among these is a large family of enzymes involved in synthesis of fatty acids, waxes, flavins, natural drugs, and antibiotics making carboncarbon bonds by use of the Claisen condensation principle. Initially, an active site nucleophile induces a transesterification by nucleophilic attack on an acylthioester substrate. In the second step, a b-carbanion thioester is generated by either proton abstraction or decarboxylation. This strong nucleophile then attacks the carbonyl carbon of the first ester, resulting in a b-keto product (Scheme I). b-Ketoacyl-acyl carrier protein (ACP) synthase {3-oxoacyl-[acyl-carrier-protein] synthase (E.C. 2.3.1.41)} I (KAS I) and KAS II from Escherichia coli represent a set of decarboxylating condensing enzymes, which we refer to as the CHH b-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 b-ketoacyl-ACP synthases, including mitochondrial b-ketoacyl-ACP synthase, bacterial plus plastid b-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 coli b-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 e , directed only towards the active site and malonyl-ACP binding area in the fatty acid complex.Abbreviations ACP, acyl carrier protein; KAS, b-ketoacyl-ACP synthase; WT-C8, KAS I-octanoyl complex.

Structural Basis for the Recognition of Mycolic Acid Precursors by KasA, a Condensing Enzyme and Drug Target from Mycobacterium Tuberculosis

“…In particular, the decarboxylation step of KAS enzymes has not been fully elucidated due to the lack of structural information to establish the location of the malonyl group. The 1,3-dioxo moiety of TLM5 is an excellent mimic of the malonyl group and, consistent with previous studies, is hydrogen-bonded to His-345 in the active site (44). However, it is not possible to distinguish between the two oxo groups (i.e.…”

“…We assume that the C 42 chain cannot be further elongated because the acyl channel is capped by the peptide backbone of residues 239 -241, thus explaining why KasA is not able to produce full-length meromycolic acids. Consistently, phospholipids mimicking the C 44 (Table 2). Furthermore, we observed that the phospholipid does not bind in an extended manner but rather contains several kinks as predefined by the geometry of the surrounding KasA cavity (Fig.…”

“…4D) of the adjacent subunit. Because all of the KasA helices mentioned above undergo conformational changes during the acyl channel open- (44). We unambiguously identified a potassium cation by analysis of the cation binding site and coordination geometry (64 -66), crystal diffraction data, and results from inductively coupled plasma optical emission spectrometry (data not shown).…”

“…S2) and were proposed to be involved in ACP-substrate binding (44), contact the terminal phosphopantetheine peptide carbonyl oxygen (Fig. 4C), which is represented by one of the TLM6 fluorines (Fig.…”

Inhibition of the fungal fatty acid synthase type I multienzyme complex

“…As in the FAS II enzymes, the active cysteine was positioned at the N-terminal end of a long ␣-helix, allowing it to exploit the helix dipole moment to increase its nucleophilicity (22). The two histidines responsible for the decarboxylation step (25,26) are similar to the FAS II KSs positioned at two loops, Ϸ3 and 5 Å apart from the nucleophilic cysteine ( Fig. 2A).…”