Mechanism-based inhibition of a mutant Escherichia coli ribonucleotide reductase (cysteine-225----serine) by its substrate CDP.

Abstract

The B1 subunit of Escherichia coli ribonucleotide reductase (EC 1.17.4.1) has been overexpressed using the pT7-5/pGP1-2 system developed by Tabor and Richardson [Tabor, S. & Richardson, C. (1985) Proc. Natl. Acad. Sci. USA 82, 1074USA 82, -1078. This method has allowed the preparation of two mutant B1 subunits in which two of the four thiols postulated to be within the active site of the enzyme, The same studies of Lin et al. (4) revealed that two additional cysteines, residues 225 and 230, are spatially close to these C-terminal cysteines. Of the seven sequences of B1 elucidated thus far, only Cys-225 of the four cysteines identified is conserved.In addition, single turnover experiments using prereduced RDPR and CDP in the absence of external reductant resulted in production of three dCDPs rather than the two anticipated from the generally accepted model of B1B2 (8). Thelander (8) also reported production of three dCDPs in a similar experiment with the first two dCDPs being produced more rapidly than the third.To account for these results, a model was proposed in which two sets of thiols are involved in catalysis per B1 protomer. Cys-225 and Cys-230 were proposed to serve as a shuttle for electrons between thioredoxin and the active site thiols, Cys-754 and Cys-759 (Fig. 1). This model predicts that four dCDPs could be produced under single-turnover conditions, and the observation of three dCDPs was rationalized as resulting from incomplete prereduction of the cysteines in B1. This model also predicts that, under single-turnover conditions, a change in Cys-754 or Cys-759 to serine would prevent dCDP production whereas a change in either Cys-225 or Cys-230 should allow production of two dCDPs (4).Studies utilizing the mechanism-based inhibitor 2'-chloro-2'-deoxyuridine 5'-diphosphate (CIUDP) also make predictions concerning the function of the redox-active thiols (9,10). If the reduction process is impaired, but the general acid catalysis function of one of the thiols is retained, then the normal substrate CDP will be converted into a mechanismbased inhibitor (Fig. 2). The postulated role of the thiols has been investigated using site-directed mutagenesis.

Cloning, sequencing, and expression of the adenosylcobalamin-dependent ribonucleotide reductase from Lactobacillus leichmannii.

“…To complete any unfinished sequences generated during the amplification, the PCR mixture was treated with Sequenase in the following manner. The reaction contained (in a final volume of 500 ul) all four dNTPs (each at 0.1 mM), lx HindlIl restriction buffer (50 mM NaCl/10 mM Tris HCl, pH 7.9/10 mM MgCl2/1 mM dithiothreitol), 10 units of Sequenase, and the PCR product. The reaction was incubated at 30°C for 30 min and stopped by the addition of EDTA to a final concentration of 10 mM.…”

“…Initial efforts focused on the pUC plasmid containing the 1.1-kb insert from the PCR. The first 60 bases read from this cloned insert encoded 20 aa (aa [8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27] of the N-terminal end of RTPR. The first 18 nt, as expected, were those that encoded the N-terminal PCR primer.…”

“…The structure of the R2 subunit of the E. coli ribonucleoside-diphosphate reductase (RDPR, EC 1.17.4.1) has been determined by crystallographic methods (8). Site-directed mutagenesis studies of conserved cysteines on the Ri subunit have begun to define residues that play an important role in catalysis (10)(11)(12)(13)(14). The unavailability of the genes has precluded these types of studies on the other classes of reductases.…”

Identification of structural domains within the large subunit of herpes simplex virus ribonucleotide reductase

“…However, the resulting complex is inactive in ribonucleotide reductase assays. Proteolysis occurs close to cysteine 582, a highly conserved residue (equivalent to cysteine 225 in E. coli R1) which is proposed to be in the active site and involved in substrate reduction (Aberg et al, 1989;Mao et al, 1989;Stubbe, 1990). Cleavage at arginine 575 may affect the active site conformation, rendering the enzyme inactive, or essential residues may be lost during additional proteolysis in this region.…”

Giant yeast cells with nonrecyclable ribonucleotide reductase

“…The reduction of one NDP by Rnr1 or Rnr3 is balanced by the formation of a disulfide bond between two cysteines of the active site (C218 and C443 in Rnr1), which inactivates the enzyme. The catalytic capacity of Rnr1 or Rnr3 is regenerated by the reduction of this disulfide bond by two C-terminal cysteines (C883 and C886 in Rnr1), which are ultimately reduced by thioredoxins or glutaredoxins (Mao et al 1989(Mao et al , 1992Camier et al 2007).…”