Enoyl-Acyl Carrier Protein Reductase (fabI) Plays a Determinant Role in Completing Cycles of Fatty Acid Elongation in Escherichia coli

Richard J Heath,Charles O Rock

doi:10.1074/jbc.270.44.26538

Abstract

The role of enoyl-acyl carrier protein (ACP) reductase (E.C. 1.3.1.9), the product of the fabI gene, was investigated in the type II, dissociated, fatty acid synthase system of Escherichia coli. All of the proteins required to catalyze one cycle of fatty acid synthesis from acetyl-CoA plus malonyl-CoA to butyryl-ACP in vitro were purified. These proteins were malonyl-CoA:ACP transacylase (fabD), beta-ketoacyl-ACP synthase III (fabH), beta-ketoacyl-ACP reductase (fabG), beta-hydroxydecanoyl-ACP dehydrase (fabA), and enoyl-ACP reductase (fabI). Unlike the other enzymes in the cycle, FabA did not efficiently convert its substrate beta-hydroxybutyryl-ACP to crotonyl-ACP, but rather the equilibrium favored formation of beta-hydroxybutyryl-ACP over crotonyl-ACP by a ratio of 9:1. The amount of butyryl-ACP formed depended on the amount of FabI protein added to the assay. Extracts from fabI(Ts) mutants accumulated beta-hydroxybutyryl-ACP, and the addition of FabI protein to the fabI(Ts) extract restored both butyryl-ACP and long-chain acyl-ACP synthesis. FabI was verified to be the only enoyl-ACP reductase required for the synthesis of fatty acids by demonstrating that purified FabI was required for the elongation of both long-chain saturated and unsaturated fatty acids. These results were corroborated by analysis of the intracellular ACP pool composition in fabI(Ts) mutants that showed beta-hydroxybutyryl-ACP and crotonyl-ACP accumulated at the nonpermissive temperature in the same ratio found in the fabI(Ts) extracts and in the in vitro reconstruction experiments that lacked FabI. We conclude that FabI is the only enoyl-ACP reductase involved in fatty acid synthesis in E. coli and that the activity of this enzyme plays a determinant role in completing cycles of fatty acid biosynthesis.

Highlights

The fatty acid synthase system of Escherichia coli is the paradigm for the type II or dissociated fatty acid synthase systems
The gel electrophoresis technique underestimated the actual amount of ␤-ketobutyryl-acyl carrier protein (ACP) formed because the high pH, temperature, and urea used to achieve the separation led to degradation and low recovery of the unstable ␤-ketoesters
Our results point to enoyl-ACP reductase as a determinant factor in completing rounds of fatty acid elongation (Fig. 1)

Summary

Introduction

The fatty acid synthase system of Escherichia coli is the paradigm for the type II or dissociated fatty acid synthase systems (for review see Ref. 1). The first step is condensation of malonyl-ACP1 with either acetyl-CoA to initiate fatty acid synthesis or the growing acyl chain to continue cycles of elongation.

Results

Conclusion