Proc. Natl. Acad. Sci. USA
Vol. 95, pp. 2723­2724, March 1998
Commentary
Ribonucleotide reductases in the twenty-first century
JoAnne Stubbe*
Departments of Chemistry and Biology, Massachusetts Institute of Technology, Cambridge, MA 02139
Ribonucleotide reductases (RNRs) catalyze the conversion of
nucleotides to deoxynucleotides in all organisms and thus play
a central role in nucleic acid metabolism (1­3). Recently,
substantive progress has been made in elucidation of the
structure (4, 5), function (6), and regulation (7) of reductases.
In addition, recently obtained sequences of reductases from
archaea and deeply rooted eubacteria (8­10) have allowed
speculation on the role of these enzymes in evolution. As
outlined below, as many questions have been raised as an-
swered, guaranteeing that reductases will be of interest to
investigate experimentally for some time to come.
One of the most amazing revelations concerning RNRs is
that despite their central role in metabolism, the essential
metal cofactors have not been evolutionarily conserved. The
RNRs have been divided into classes on the basis of these
observations (Fig. 1). The class I and class II RNRs, requiring
a diferric­tyrosyl radical (11) and adenosylcobalamin
(AdoCbl) (12, 13), respectively, have been most extensively
studied. From these studies and more recent studies on the
intriguing RNR utilizing an essential glycyl radical cofactor
(class III) found only in obligate anaerobes (14), a general
mechanistic paradigm has evolved (15, 16) (Fig. 1). Whether
the class IV RNR, containing a putative manganese cluster­
tyrosyl radical cofactor (17), is in accord with this paradigm
remains to be established.
The model is that, despite the diversity of cofactors and the
enzymes' primary and quaternary structures, the mechanism of
nucleotide reduction, involving complex and exquisitely con-
trolled radical-dependent redox chemistry, has been conserved
(16). The function of the diverse metallo-cofactors, as first
proposed in the 1960s, is to initiate some type of radical-
dependent reduction process. This model has grown more so-
phisticated and complex, because of the availability of data from
physical organic studies on these enzymes. In addition, multiple
sequences from the class I, II, and III RNRs and the resulting
accessibility of large amounts of these proteins have facilitated
examination in detail of the function of the cofactors (Fig. 1).
These studies collectively have allowed further definition of the
role of each cofactor as a generator of an essential thiyl radical
that initiates the nucleotide reduction process by 3 -hydrogen
abstraction from the nucleotide (6, 18, 19).
Support for this postulate has recently been provided from
studies on both the structure and function of class I and class II
RNRs. Eklund and his collaborators have reported the first
structure of each of the two subunits of the E. coli class I RNR:
R1 (4, 7) and R2 (20, 21). R2 contains the diferric­tyrosyl radical
cofactor and R1 contains the active site, including the essential
cysteine designated to become a thiyl radical. A cartoon of the
active site of this RNR (R1) is also shown in Fig. 1, where the
three required cysteines (the thiyl radical and the two cysteines
delivering the reducing equivalents) are housed in a 10-stranded
, -barrel with a loop penetrating the center of this structure
containing at its tip the precursor to the thiyl radical. The
remarkable congruency in the chemistry observed with the active
site mutants of class I and II RNRs and a variety of mechanism-
based inhibitors of class I, II, and III RNRs (22) strongly suggest
that the active site of the class I enzyme will serve as a paradigm
for the active site structure that will be found in the other classes
of RNRs. Recent rapid freeze quench EPR spectroscopy has
provided the first direct evidence with class II enzymes that the
metallo-cofactor generates, in a kinetically competent fashion, a
thiyl radical that is directly involved in nucleotide reduction (6).
The chemical similarities in all classes of RNRs thus suggest that
this will be a common theme (Fig. 1).
Support for the importance of thiyl radicals in catalysis is
provided for class I RNRs indirectly by sequence studies of
RNRs from Thermoplasma acidophila by Tauer and Benner (8)
and the more recent studies on other organisms of Riera et al.
(9) and Jordan et al. (10). These reductases use AdoCbl as a
cofactor (class II), but, strikingly, their active sites are homol-
ogous to those of the class I RNRs. Recent rapid freeze quench
 1998 by The National Academy of Sciences 0027-8424 98 952723-2$2.00 0
PNAS is available online at http: www.pnas.org.
The companions to this commentary are published on pages 53, 475,
and 13487 of volume 94.
*To whom reprint requests should be addressed. e-mail: stubbe@
mit.edu.
FIG. 1. The active site of the Escherichia coli R1 subunit, adapted from
Uhlin and Eklund (4). Cys-439, the site of the putative thiyl radical, is at
the tip of the central loop of the barrel with Glu-441, the conserved
glutamate adjacent to this residue. In addition, a disulfide is shown
between Cys-225 and Cys-419, the site of delivery of the reducing
equivalents to make deoxynucleotide. Each class of RNR thus far
characterized has a different cofactor: the class I RNR has a diferric­
tyrosyl radical (blue balls represent the orbital with the unpaired elec-
tron); class II utilizes AdoCbl; class III utilizes S-adenosylmethionine and
an iron­sulfur cluster to generate a glycyl radical; and class IV is proposed
to have a Mn cofactor adjacent to a tyrosyl radical. The cofactors' function
is proposed to be the generation of the thiyl radical, which will be located,
regardless of the class of RNR, in an active site with secondary and
tertiary structure similar to that observed in class I R1. The figure was
generated by using MOLSCRIPT (34) and RASTER3D (35).
2723
EPR studies on the T. acidophila RNR (23) suggest that it also
generates a thiyl radical in a kinetically competent fashion.
These recent sequence data in conjunction with physical
methods thus support the proposal that the metallo-cofactors
generate thiyl radicals in a nucleotide-binding domain that will
share common secondary and tertiary structural features.
Further support for the model that all classes of RNRs share
a common secondary and tertiary nucleotide reduction domain
has been proposed on the basis of the observation of gross
similarities in the complex regulation of the selectivity of the
nucleotide reduction process in the class I, II, and III RNRs (2,
24). The appropriate deoxynucleotide (dNTP) governs which
nucleotide is reduced, that is, the specificity of nucleotide reduc-
tion. The regulation argument has thus been used as an evolu-
tionary determinant (24). However, the structure of E. coli R1
(composed of two identical protomers) suggests that the speci-
ficity site is at the R1 dimer interface. Furthermore, the studies
to date suggest that the Lactobacillus leichmannii and T. acidophi-
lus RNRs are monomers. Thus it is unlikely that the structural
similarities in regulation have been evolutionarily conserved,
unless the active unit of the class I RNRs is actually a protomer
of R1, or that as yet undetected dimers are the catalytically active
species in the L. leichmannii and T. acidophilus RNRs.
The recent structure of R1 has better defined the site that
governs specificity of reduction, and in this class I RNR a
second allosteric site that governs turnover rate (7). This
second site does not appear to have been evolutionarily
conserved. This initial structural glimpse has provided a
starting point for thinking about the amazing complexity of the
specificity of the reduction process essential for the fidelity of
DNA replication.
An added layer of regulatory complexity is apparent from
recent studies in Saccharomyces cerevisiae that have revealed two
class I RNRs (both R1 and R2 subunits) with a high degree of
sequence homology, proposed to be involved in replication and
repair (25­27). One of these RNRs is dramatically inducible by
DNA-damaging agents and is clearly involved in a complex signal
transduction cascade in some way controlling the cell cycle (28).
Whether two such RNRs exist in humans remains to be estab-
lished. These questions are central to understanding nucleic acid
metabolism They take on a new light given the recent approval
of the Food and Drug Administration of gemcitabine (29). This
nucleoside, subsequent to its phosphorylation to the diphosphate,
is a stoichiometric inactivator of reductase. Decreased dNDP
pools are thought to lead to decreased dNTP pools, a situation
that facilitates the incorporation of gemcitabine 5 -triphosphate
into nucleic acid by DNA polymerase, where it functions as a
chain terminator. This synergism is thought to provide an expla-
nation for the observed cytotoxicity and therapeutic efficacy of
the drug (ref. 30; W. A. van der Donk, G. Yu, L. Perez, R. J.
Sanchez, and J.S., unpublished work).
Studies in yeast and recent studies in mammalian cell culture
(31­33) raise additional provocative questions central to under-
standing nucleic acid metabolism. Is it possible that assembly and
disassembly of the diferric­tyrosyl radical cofactor is intimately
involved in regulation of the nucleotide reduction process? This
proposition would require a link between nucleic acid and energy
metabolism as one electron is required for reduction of the
essential tyrosyl radical and one electron is required for its
regeneration from diferrous R2. Recent studies in yeast defining
the gene products responsible for iron and copper homeostasis
raise the intriguing possibility that this question will be answerable.
Thus the past five years have solidified in a gratifying way our
understanding of the mechanism of nucleotide reduction and the
prominent role of the metallo-cofactors in this process. They have
also raised thought-provoking questions for the class I RNRs
regarding the regulation of nucleotide reduction in replication
and repair in vivo at the level of allosteric feedback and cofactor
formation and destruction, as well as the prospect of reductase
compartmentalization. Thus the future studies on this exciting
system appear to be as intriguing as those of the past.
Thanks to all of the Stubbe group members who have worked on this
project, thanks to Joe Kappock for preparation of Fig. 1, and thanks
to Novartis for support of the Stubbe group. Our work on ribonucle-
otide has been generously supported over the years by National
Institutes of Health Grant GM29595.
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formation, yet this gene product appears to be essential (25).
2724 Commentary: Stubbe Proc. Natl. Acad. Sci. USA 95 (1998)