Tài liệu Báo cáo khoa học: The kinetic properties of various R258 mutants of deacetoxycephalosporin

The kinetic properties of various R258 mutants

of deacetoxycephalosporin C synthase

Hwei-Jen Lee1

, Young-Fung Dai1

, Chia-Yang Shiau2

, Christopher J. Schofield3 and Matthew D. Lloyd4

1

Department of Biochemistry, National Defense Medical Centre, Taipei, Taiwan, ROC; 2

Institute of Medical Science, National

Defense Medical Centre, Taipei, Taiwan, ROC; 3

The Oxford Centre for Molecular Sciences and Dyson Perrins Laboratory,

South Parks Road, Oxford, UK; 4

Department of Pharmacy and Pharmacology, University of Bath, Claverton Down, Bath, UK

Site-directed mutagenesis was used to investigate the control

of 2-oxoacid cosubstrate selectivity by deacetoxycephalo￾sporin C synthase. The wild-type enzyme has a requirement

for 2-oxoglutarate and cannot efficiently use hydrophobic

2-oxoacids (e.g. 2-oxohexanoic acid, 2-oxo-4-methyl-penta￾noic acid) as the cosubstrate. The following mutant enzymes

were produced: R258A, R258L, R258F, R258H and

R258K. All of the mutants have broadened cosubstrate

selectivity and were able to utilize hydrophobic 2-oxoacids.

The efficiency of 2-oxoglutarate utilization by all mutants

was decreased as compared to the wild-type enzyme, and

in some cases activity was abolished with the natural

cosubstrate.

Keywords: b-lactam biosynthesis; cephem; chemical cosub￾strate rescue; nonhaem iron(II) oxygenase; 2-oxoglutarate.

Deacetoxycephalosporin C synthase (DAOCS; Swiss-Prot

P18548) catalyses a key step in the cephamycin C biosyn￾thetic pathway in Streptomyces clavuligerus, i.e. the ring￾expansion of penicillin N (1) to deacetoxycephalosporin C

(DAOC, 2) [1–7] (Scheme 1). A sequence-related oxygenase,

deacetylcephalosporin C synthase (DACS; Swiss-Prot

42220), catalyses the subsequent hydroxylation of the exocy￾clic methyl group of (2) to give deacetylcephalosporin C

(DAC, 3). The DAC product is then converted by a series of

reactions, including 7-hydroxylation, into cephamycin C (4).

Oxidative reactions in this pathway are catalysed by

nonhaem iron(II) and 2-oxoglutarate (2-OG)-dependent

oxygenases [4]. Two of these enzymes, DAOCS and DACS,

are part of a sequence-related subgroup of enzymes [8],

which also include deacetoxy/deacetylcephalosporin C syn￾thase (the Cephalosporium acremonium bifunctional protein

catalysing the both ring-expansion and hydroxylation

reactions; Swiss-Prot P11935) and isopenicillin N synthase

(which is not a 2-OG-dependent oxygenase, but catalyses

the formation of the bicyclic penicillin nucleus; S. clavuli￾gerus enzyme Swiss-Prot P10621).

Understanding of the catalytic mechanism of DAOCS

[9–11] has been advanced by a combination of biochemical

and X-ray crystallographic studies [1,12–17]. These studies

have given insights into how the oxidation of 2-oxoglutarate

(the cosubstrate) and the penicillin substrate are coupled.

R258 and S260 are located in the 2-OG binding pocket and

bind the 5-carboxylate of the cosubstrate by electrostatic

interactions. Mutation of R258 to glutamine results in loss

of coupling between penicillin oxidation and 2-OG conver￾sion [17]. Moreover, the activity of the R258Q mutant could

be restored by the use of
unnatural 2-oxoacids (which are

not utilized by the wild-type enzyme) as cosubstrates

(
chemical cosubstrate rescue) [17]. A similar change in

cosubstrate selectivity upon mutation of the analogous

arginine residue has been observed with the 2-OG-depend￾ent oxygenase, phytanoyl-CoA 2-hydroxylase [18,19]. This

paper reports the biochemical properties of other DAOCS

mutants, in which R258 has been replaced with uncharged

or positively charged amino acids.

Materials and methods

Materials

Chemicals were obtained from the Sigma-Aldrich Chemical

Co. or Merck and were of at least analytical grade or higher.

Reagents were also supplied by: Amersham Biosciences

(protein chromatography systems and columns); Bohringer￾Mannheim (ATP); MBI (1 kb and 100 bp DNA gel

markers); Bio-Rad (Kunkel mutagenesis reagents); New

England Bio-Laboratories (enzymes for molecular biology);

Novagen (pET vectors); Gibco/BRL (mutagenesis primers);

Phenomenex (HPLC columns).

Correspondence to H.-J. Lee, Department of Biochemistry, National

Defense Medical Centre, No. 161, Sec. 6, Minchuan East Road,

Neihu 114, Taipei, Taiwan, Republic of China.

Fax: + 886 2 87921544, Tel.: + 886 2 87910832,

E-mail: [email protected]; and

M. D. Lloyd, Department of Pharmacy and Pharmacology,

University of Bath, Claverton Down, Bath BA2 7AY.

Tel.: + 44 1225 386786,

E-mail: [email protected]

Abbreviations: DACS, deacetylcephalosporin C synthase; DAOCS,

deacetoxycephalosporin C synthase; ESI-MS, electrospray ionization

mass spectrometry; G-7-ADCA, phenylacetyl-7-aminodeacetoxy

cephalosporanic acid; 2-OG, 2-oxoglutarate; 2-OH, 2-oxohexanoate;

2-OMP, 2-oxo-4-methyl-pentanoate.

(Received 14 November 2002, revised 29 January 2003,

accepted 4 February 2003)

Eur. J. Biochem. 270, 1301–1307 (2003)
FEBS 2003 doi:10.1046/j.1432-1033.2003.03500.x