Enzyme Boasts a Radical Makeover

The bacterial pathogen chlamydia trachomatis contains an enzyme called ribonucleotide reductase that's essential for making and repairing DNA. But the pathogen's version of this enzyme is strikingly different from those found in other organisms, according to Swedish researchers. They hypothesize that the enzyme's unique features might help this and related pathogens evade the chemical defenses of the pathogens' hosts.

DNA is assembled from four deoxyribonucleotide building blocks. Ribonucleotide reductases (RNRs) synthesize these deoxyribonucleotides by replacing a hydroxyl group on the ribose ring of the ribonucleotide precursors with hydrogen. All RNRs use a cysteine thiyl radical to do this chemistry, but they don't all generate the radical in the same way: The most common type of RNR--found in humans as well as Escherichia coli and other bacteria--uses a diiron cofactor to make a stable tyrosine radical, which in turn generates the cysteine thiyl radical. Other organisms contain RNRs that use a glycyl radical or a cobalt-containing cofactor to make the cysteine thiyl radical.

The sequence of chlamydial RNR is homologous to human and E. coli RNRs. But the pathogenic enzyme lacks the conserved radical-harboring tyrosine that characterizes these enzymes. Scientists had assumed that a nearby tyrosine would substitute, because mutating this residue inactivates chlamydial RNR. But the enzyme's X-ray crystal structure--solved to 1.7-Å resolution by Pär Nordlund, Martin Högbom, and Pål Stenmark of Stockholm University and their coworkers--reveals that the enzyme lacks a tyrosine radical site entirely [Science, 305, 245 (2004)].

Instead, a phenylalanine residue occupies the spot where the tyrosine radical was expected to be. The tyrosine that scientists suspected might serve as a stand-in points out into solution. This tyrosine's position "excludes it as a candidate for harboring a stable radical," Stenmark says.

So how does this RNR generate the required cysteine thiyl radical? Nordlund's team suggests that instead of using its diiron cofactor to make a tyrosine radical that in turn generates the cysteine thiyl radical, the chlamydial enzyme uses an iron-centered radical to generate the cysteine thiyl radical directly. The team uses electron paramagnetic resonance spectroscopy to show that an iron-centered radical is in fact present in the reconstituted enzyme.

NOTABLY, the diiron center in chlamydial RNR is markedly different from that found in its human and bacterial relatives. These RNRs use three glutamates and one aspartate to coordinate the two iron atoms, which are bridged by an oxo group and also bind two terminal water molecules. But the chlamydial enzyme's diiron cofactor more closely resembles that of diiron monoxygenases, using two histidines and four glutamates to hold onto its iron atoms. The irons appear to be bridged by two hydroxide groups and to bind one terminal water molecule, Nordlund's team reports.

C. trachomatis isn't likely to be the only organism that contains this novel type of RNR. Nordlund and his coworkers have identified about half a dozen RNRs that lack the radical-harboring tyrosine, including ones from human and animal pathogens. They hypothesize that these organisms might have adopted the unusual RNR to survive under certain conditions. Mammalian immune systems often release nitric oxide to destroy invading pathogens--and nitric oxide can destroy tyrosine radicals. So to evade their hosts' chemical defenses, C. trachomatis and other pathogens might have turned to an RNR that doesn't require a tyrosine radical, Stenmark and Högbom suggest.

"Their proposal is provocative," notes chemistry and biology professor JoAnne Stubbe of Massachusetts Institute of Technology, but remains to be experimentally tested.