Tài liệu Báo cáo khoa học: Regulation of dCTP deaminase from Escherichia coli by nonallosteric dTTP

Regulation of dCTP deaminase from Escherichia coli by

nonallosteric dTTP binding to an inactive form of the

enzyme

Eva Johansson1,2, Majbritt Thymark1

, Julie H. Bynck1

, Mathias Fanø3

, Sine Larsen1,2

and Martin Willemoe¨ s3

1 Centre for Crystallographic Studies, Department of Chemistry, University of Copenhagen, Denmark

2 European Synchrotron Radiation Facility, Grenoble, France

3 Department of Molecular Biology, University of Copenhagen, Denmark

Synthesis of dTMP by thymidylate synthase proceeds

by the reductive methylation of dUMP, which is

obtained via one of two parallel pathways. One path￾way, considered to be a minor supplier of dTTP [1–3],

involves the reduction of UDP (UTP) by the action of

ribonucleotide reductase. Subsequently, dUDP is phos￾phorylated to dUTP and cleaved to dUMP. The main

supply of dUMP, however, involves the deamination

Keywords

deoxynucleotide metabolism; dUTP; enzyme

regulation; hysteresis; deamination

Correspondence

E. Johansson, Diabetes Protein Engineering,

Novo Nordisk A ⁄ S, Novo Nordisk Park,

DK-2760 Ma˚løv, Denmark

Fax: +45 4444 4256

Tel: +45 4442 1189

E-mail: [email protected]

or

M. Willemoe¨ s, Department of Molecular

Biology, University of Copenhagen, Ole

Maaløes vej 5, DK-2200 Copenhagen N,

Denmark

Fax: +45 3532 2128

Tel: +45 3532 2030

E-mail: [email protected]

Database

The atomic coordinates and structure fac￾tors have been deposited in the Protein

Data Bank with the PDB ID codes 2j4q

(E138:dTTP) and 2j4 h (H121A:dCTP) and

can be accessed at http://www.rcsb.org

(Received 12 April 2007, revised 12 June

2007, accepted 18 June 2007)

doi:10.1111/j.1742-4658.2007.05945.x

The trimeric dCTP deaminase produces dUTP that is hydrolysed to dUMP

by the structurally closely related dUTPase. This pathway provides

70–80% of the total dUMP as a precursor for dTTP. Accordingly, dCTP

deaminase is regulated by dTTP, which increases the substrate concentra￾tion for half-maximal activity and the cooperativity of dCTP saturation.

Likewise, increasing concentrations of dCTP increase the cooperativity of

dTTP inhibition. Previous structural studies showed that the complexes of

inactive mutant protein, E138A, with dUTP or dCTP bound, and wild-type

enzyme with dUTP bound were all highly similar and characterized by hav￾ing an ordered C-terminal. When comparing with a new structure in which

dTTP is bound to the active site of E138A, the region between Val120 and

His125 was found to be in a new conformation. This and the previous con￾formation were mutually exclusive within the trimer. Also, the dCTP com￾plex of the inactive H121A was found to have residues 120–125 in this new

conformation, indicating that it renders the enzyme inactive. The C-ter￾minal fold was found to be disordered for both new complexes. We suggest

that the cooperative kinetics are imposed by a dTTP-dependent lag of

product formation observed in presteady-state kinetics. This lag may be

derived from a slow equilibration between an inactive and an active confor￾mation of dCTP deaminase represented by the dTTP complex and the

dUTP ⁄ dCTP complex, respectively. The dCTP deaminase then resembles a

simple concerted system subjected to effector binding, but without the use

of an allosteric site.

Abbreviations

E138A, mutant dCTP deaminase with a Glu138 to Ala substitution; H121A, mutant dCTP deaminase with a His121 to Ala substitution;

V122G, mutant dCTP deaminase with a Val122 to Gly substitution.

4188 FEBS Journal 274 (2007) 4188–4198 ª 2007 The Authors Journal compilation ª 2007 FEBS