Tài liệu Báo cáo khoa học: Fatty acid synthesis Role of active site histidines and lysine in

Fatty acid synthesis

Role of active site histidines and lysine in Cys-His-His-type

b-ketoacyl-acyl carrier protein synthases

Penny von Wettstein-Knowles1

, Johan G. Olsen2

, Kirsten A. McGuire1 and Anette Henriksen2

1 Genetics Department, Molecular Biology and Physiology Institute, Copenhagen University, Denmark

2 Biostructure Group, Carlsberg Laboratory, Copenhagen, Denmark

The formation of carbon–carbon bonds is a funda￾mental 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 carbon–

carbon bonds by use of the Claisen condensation prin￾ciple. Initially, an active site nucleophile induces a

transesterification by nucleophilic attack on an acyl￾thioester 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-pro￾tein] 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

Keywords

active site mutations; condensation reaction;

fatty acid synthase; reaction mechanism;

b-ketoacyl-ACP synthase

Correspondence

P. von Wettstein-Knowles, Genetics

Department, Molecular Biology and

Physiology Institute, Copenhagen University,

Øster Farimagsgade 2A, DK-1353

Copenhagen, Denmark

Fax: +45 35322113

Tel: +45 35322180

E-mail: [email protected]

A. Henriksen, Carlsberg Laboratory,

Biostructure Group, Gamle Carlsberg Vej 10,

DK-2500 Valby, Denmark

Fax: +45 33274708

Tel: +45 33275222

E-mail: [email protected]

(Received 10 August 2005, revised 2

December 2005, accepted 12 December

2005)

doi:10.1111/j.1742-4658.2005.05101.x

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 pri￾mer 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 mitochond￾rial 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 construc￾ted and their ability to carry out the trans thioesterification, decarboxyla￾tion 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 Ne, 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.

FEBS Journal 273 (2006) 695–710 ª 2006 The Authors Journal compilation ª 2006 FEBS 695