Tài liệu Báo cáo Y học: Studies into factors contributing to substrate specificity of membrane-bound

Studies into factors contributing to substrate specificity

of membrane-bound 3-ketoacyl-CoA synthases

Brenda J. Blacklock and Jan G. Jaworski

Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio, USA

We are interested in constructing a model for the substrate￾binding site of fatty acid elongase-1 3-ketoacyl CoA synthase

(FAE1 KCS),the enzyme responsible for production of very

long chain fatty acids of plant seed oils. Arabidopsis thaliana

and Brassica napus FAE1 KCS enzymes are highly homo￾logous but the seed oil content of these plants suggests that

their substrate specificities differ with respect to acyl chain

length. We used in vivo and in vitro assays of Saccharomyces

cerevisiae-expressed FAE1 KCSs to demonstrate that the

B. napus FAE1 KCS enzyme favors longer chain acyl sub￾strates than the A. thaliana enzyme. Domains/residues

responsible for substrate specificity were investigated by

determining catalytic activity and substrate specificity of

chimeric enzymes of A. thaliana and B. napus FAE1 KCS.

The N-terminal region,excluding the transmembrane

domain,was shown to be involved in substrate specificity.

One chimeric enzyme that included A. thaliana sequence

from the N terminus to residue 114 and B. napus sequence

from residue 115 to the C terminus had substrate specificity

similar to that of A. thalianaFAE1 KCS. However,a K92R

substitutionin this chimeric enzyme changed the specificity to

that of the B. napus enzyme without loss of catalytic activity.

Thus,this study was successful in identifying a domain

involved in determining substrate specificity in FAE1 KCS

and in engineering an enzyme with novel activity.

Keywords: Arabidopsis thaliana; Brassica napus; fatty acid

elongation; 3-ketoacyl-CoA synthase.

The very long chain fatty acids (VLCFA) found in seed

oils are derived from the elongation of products of de novo

fatty acid biosynthesis [1]. The initial reaction of elonga￾tion,i.e. the iterative condensation of acyl units with

malonyl-CoA,is catalyzed in the seed by the membrane￾bound fatty acid elongase-1 3-ketoacyl-CoA synthase

(FAE1 KCS) [2]. Subsequent reduction and dehydration

reactions are carried out by distinct and separate enzymes

that are just beginning to be characterized [1,3,4].

FAE1 KCS was first identified in Arabidopsis thaliana [5]

and homologues have been found in oleaginous species

such as Brassica napus, B. juncea,and Simmondsia chinen￾sis [6–10]. The functional similarity among these enzymes

is demonstrated by the ability of the jojoba FAE1 KCS to

complement the canola fatty acid elongation mutation

even though jojoba produces wax rather than triacylgly￾cerol,as found in other seed oils [6].

Examination of the VLCFA content of the seed oils of

A. thaliana and B. napus reveals differences in the levels of

eicosenoic (20:1) and erucic (22:1) fatty acids. In A. thaliana

seed oil,20% of the total fatty acids are VLCFA of which

18% of the total fatty acids are in the form of 20:1 and 2%

in the form of 22:1 [11]. In B. napus seed oil,62% of the

total fatty acids are monounsaturated VLCFA,10% as 20:1

and 52% as 22:1 [12]. As FAE1 KCS is responsible for

VLCFA production in oilseeds [2,5,6], this diversity in

VLCFA content suggests that A. thaliana and B. napus

FAE1 KCS enzymes have distinct substrate specificities

with the B. napus enzyme favoring longer chain length

substrates than the A. thaliana enzyme. The high sequence

identity (86%) between these two enzymes further suggests

that the determinants responsible for fatty acid substrate

specificity in FAE1 KCS are few and potentially identifi￾able.

The significant amino acid sequence homology of

FAE1 KCSs with soluble condensing enzymes,such as

chalcone synthase and 3-ketoacyl-acyl carrier protein

synthases (KASs) is consistent with a role for FAE1 KCSs

as fatty acid condensing enzymes [5,6,13]. Our understand￾ing of the structure/function relationships of soluble

condensing enzymes has been greatly advanced with the

recent crystal structures of KAS I,-II,and -III and chalcone

synthase [14–19]. However,only limited information is

available about the structure of the membrane-bound

KCSs. Secondary structural analysis of the family of

FAE1 KCS enzymes reveals two putative transmembrane

domains at the N termini of the proteins. Recent work in

our laboratory has confirmed that the amino terminus of

Arabidopsis FAE1 KCS is involved in anchoring the

enzyme to the membrane [20]. The difficulty inherent in

crystallizing membrane-bound enzymes required us to take

a different approach to probing the structure/function

relationships of FAE1 KCS. Here,we report utilization of a

domain-swapping approach to investigate the structural

domains and residues responsible for substrate specificity in

FAE1 KCS.

Correspondence to: B. J. Blacklock,Department of Chemistry and

Biochemistry,Miami University,Oxford,OH 45056,USA.

Fax: +1 513 529 5715,Tel.: +1 513 529 1641,

E-mail: [email protected]

Abbreviations: VLCFA,very long chain fatty acid; FAE1 KCS,fatty

acid elongase-1 3-ketoacyl CoA synthase; KAS,3-ketoacyl-acyl

carrier protein synthase; cm-ura,complete minimal dropout media

lacking uracil; FAME,fatty acid methyl ester.

Note: The SWISS-PROT accession numbers for the FAE1 KCS are:

Arabidopsis thaliana,Q38860; Brassica napus,O23738.

(Received 31 May 2002,revised 2 August 2002,

accepted 12 August 2002)

Eur. J. Biochem. 269,4789–4798 (2002)
FEBS 2002 doi:10.1046/j.1432-1033.2002.03176.x