Tài liệu Báo cáo khóa học: Active-site residues and amino acid specificity of the bacterial

Active-site residues and amino acid specificity of the bacterial

4¢-phosphopantothenoylcysteine synthetase CoaB

Thomas Kupke

Lehrstuhl fu¨r Mikrobielle Genetik, Universita¨t Tu¨bingen, Tu¨bingen, Germany

In bacteria, coenzyme A is synthesized in five steps from

D-pantothenate. The Dfp flavoprotein catalyzes the synthe￾sis of the coenzyme A precursor 4¢-phosphopantetheine

from 4¢-phosphopantothenate and cysteine using the cofac￾tors CTP and flavine mononucleotide via the phospho￾peptide-like compound 4¢-phosphopantothenoylcysteine.

The synthesis of 4¢-phosphopantothenoylcysteine is cata￾lyzed by the C-terminal CoaB domain of Dfp and occurs via

the acyl-cytidylate intermediate 4¢-phosphopantothenoyl￾CMP in two half reactions. In this new study, the molecular

characterization of the CoaB domain is continued. In addi￾tion to the recently described residue Asn210, two more

active-site residues, Arg206 and Ala276, were identified

and shown to be involved in the second half reaction of

the (R)-4¢-phospho-N-pantothenoylcysteine synthetase. The

proposed intermediate of the (R)-4¢-phospho-N-panto￾thenoylcysteine synthetase reaction, 4¢-phosphopantothe￾noyl-CMP, was characterized by MALDI-TOF MS and it

was shown that the intermediate is copurified with the

mutant His-CoaB N210H/K proteins. Therefore, His-CoaB

N210H and His-CoaB N210K will be of interest to elucidate

the crystal structure of CoaB complexed with the reaction

intermediate. Wild-type His-CoaB is not absolutely specific

for cysteine and can couple derivatives of cysteine to

4¢-phosphopantothenate. However, no phosphopeptide-like

structure is formed with serine. Molecular characterization

of the temperature-sensitive Escherichia coli dfp-1 mutant

revealed that the residue adjacent to Ala276, Ala275 of the

strictly conserved AAVAD(275–279) motif, is exchanged

for Thr.

Keywords: coenzyme A biosynthesis; 4¢-phosphopantethe￾ine; 4¢-phosphopantothenoylcysteine synthetase; Dfp flavo￾protein; cysteine metabolism.

4¢-Phosphopantetheine (PP) coenzymes such as coen￾zyme A are the biochemically active forms of the vitamin

pantothenic acid. In coenzyme A, 4¢-phosphopantetheine

is covalently linked to an adenylyl group, whereas it is

covalently linked to a serine hydroxyl group in acyl carrier

proteins. 4¢-Phosphopantetheine is also cofactor of

enzymes that catalyze the biosynthesis of polypeptide

antibiotics [1]. Lipmann discovered and characterized

coenzyme A [2] and Lynen elucidated that the thiol

group of the cysteamine moiety of coenzyme A is the

functional group by activating substrates as thioesters [3].

In Escherichia coli and most eubacteria, the synthesis of

4¢-phosphopantetheine, which is also the key reaction in

coenzyme A biosynthesis, is catalyzed from 4¢-phospho￾pantothenate and cysteine by the bifunctional Dfp (CoaBC)

flavoproteins in a multistep process (Fig. 1; [4–7]). In the

first step, 4¢-phosphopantothenate is activated by reaction

with CTP. The 4¢-phosphopantothenoyl-cytidylate formed

is attacked by cysteine and 4¢-phosphopantothenoylcysteine

(PPC) is synthesized. These reactions occur at the

C-terminal CoaB domain of Dfp. The next step is the

FMN-dependent oxidative decarboxylation of PPCto

4¢-phosphopantothenoylaminoethenethiol, which is then

reduced to 4¢-phosphopantetheine; both partial reactions

are catalyzed by the N-terminal CoaC domain. Oxidative

decarboxylation of peptidyl-cysteines had already been

detected before as important step in the biosynthesis of the

lantibiotics epidermin and mersacidin catalyzed by the

LanD enzymes EpiD and MrsD, respectively [8–10]. Flavin￾dependent oxidative decarboxylation of PPCas an initial

step in the conversion of PPCto PP had been proposed by

Kupke et al. in 2000 [6] and was later confirmed for the

plant PPCdecarboxylase AtHAL3a (AtCoaC) by purifying

oxidatively decarboxylated pantothenoylcysteine as a reac￾tion intermediate [11] and by determining the crystal

structure of AtHAL3a C175S complexed with this enethiol

intermediate [12]. Dfp, AtHAL3a, EpiD and MrsD belong

to a new family of flavoproteins that was named HFCD

(homo-oligomeric flavin-containing Cys decarboxylases)

[6,13].

In a recently published study [5], the PPC-synthetase

activity of the CoaB domain of Dfp was shown, a

dimerization motif within CoaB was proposed and the

strictly conserved residues N210 and K289 were prelimin￾ary investigated with respect to their ability to synthesize

PPCand the 4¢-phosphopantothenoyl-CMP intermediate.

Here, the molecular characterization of the bacterial PPC

Correspondence to T. Kupke, Lehrstuhl fu¨r Mikrobielle Genetik,

Universita¨t Tu¨bingen, Auf der Morgenstelle 15, Verfu¨gungsgeba¨ude,

72076 Tu¨bingen, Germany.

Fax: + 49 7071 295937, Tel.: + 49 7071 2977608,

E-mail: [email protected]

Abbreviations: His-CoaA, MRGSHHHHHHGSML-CoaA; His￾CoaB, MRGSHHHHHHG-Dfp S–R(181–406); IPTG, isopropyl

thio-b-D-galactoside; PP, 4¢-phosphopantetheine; PPC,

(R)-4¢-phospho-N-pantothenoylcysteine.

(Received 2 October 2003, accepted 11 November 2003)

Eur. J. Biochem. 271, 163–172 (2004) FEBS 2003 doi:10.1046/j.1432-1033.2003.03916.x