63WWW.CEN-ONLINE.ORG SEPTEMBER 6, 2010

ACCURATELY DETERMINING which met-als a given metalloenzyme uses in its daily work can be tricky business, with in vitro experiments not always reflecting what truly happens inside cells. At the Ameri-can Chemical Society national meeting in Boston, chemists told a cautionary tale: By taking a closer look at the machinery that bacterial cells use to build the active site of a ribonucleotide reductase, these research-ers have redefined which metal makes this enzyme tick.

Found in all organisms, ribonucleotide reductases (RNRs) are essential for mak-ing and repairing DNA. They supply the deoxyribonucleotides that make up DNA

by using a cysteine thiyl radical to initiate replacement of a hydroxyl on the ribose ring of the corresponding ribonucleotide with hydrogen.

Escherichia coli and some other patho-genic bacteria make two different kinds of RNRs. Both were long thought to rely on a diiron cofactor to generate a stable tyrosine radical that in turn creates the catalytic cysteine thiyl radical.

But one of these putative diiron enzymes is actually a dimanganese enzyme, reported enzymologist JoAnne Stubbe of Massachu-setts Institute of Technology, in a session sponsored by the Division of Biological Chemistry. She and grad student Joseph

A. Cotruvo Jr. published spectroscopic and bio-chemical evidence for the existence of the dimanga-nese RNR in E. coli earlier this year ( Biochemistry 2010, 49, 1297).

Being able to make a manganese-dependent RNR may come in handy when pathogenic bac-teria encounter chal-lenging conditions—for example, when they are camped out in a human host, where available iron is scarce and oxi-

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INJECTION SITE NrdI (green) injects a peroxide species generated from O2 at its flavin cofactor (yellow) via a tunnel (blue mesh) into the RNR’s active site. There, the peroxide species oxidizes the enzyme’s Mn 2 cofactor (purple spheres) which in turn creates a stable radical on a nearby tyrosine (pink) and activates the enzyme.

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64WWW.CEN-ONLINE.ORG SEPTEMBER 6, 2010

dative threats abound. Under such conditions, they may well scuttle their normal diiron RNR in favor of a dimanganese RNR, Stubbe said.

Indeed, Cotruvo has managed to isolate active dimanganese RNR from a strain of E. coli lacking the machinery needed to take up iron, she reported. And last month, a Ger-man team published spectroscopic and crystallographic evidence that Corynebacterium ammoniagenes, a bacterium that makes its home in the human intestine, also makes a diman-ganese RNR ( J. Am. Chem. Soc., DOI: 10.1021/ja1036995).

Relying on manganese instead of iron comes at a cost. All that E. coli’ s diiron RNR requires to generate the critical thiyl radical is O 2 , Stubbe not-ed. The O 2 oxidizes the diiron center, which then generates a stable tyrosine radical that in turn creates the catalytic thiyl radical. But “unlike iron, manga-nese cannot be oxidized by O 2 ,” she added. As a consequence, E. coli must supply its di-manganese RNR with an accessory protein that can safely deliver a stronger oxidant.

ENZYMOLOGIST J. Martin Bollinger Jr. of Pennsylvania State University likens this accessory protein, dubbed NrdI, to a surgeon. NrdI docks to the enzyme and carefully injects a peroxide species, an oxi-dant capable of oxidizing the dimanganese active site and generating the amino acid radicals required for deoxyribonucleotide synthesis.

Stubbe and Cotruvo recently teamed up with Northwestern University crystal-lographer Amy C. Rosenzweig and postdoc Amie K. Boal to probe how NrdI manages this feat. At the ACS meeting, Rosenzweig described the team’s efforts to obtain structures of the manganese RNR bound to NrdI. With these structures, the research-ers were able to pinpoint within the pro-tein-protein complex the location of the flavin cofactor that NrdI uses to produce the peroxide species from O 2 . The work was published in Science earlier this month (DOI: 10.1126/science.1190187).

The researchers even managed to map the tunnel through which NrdI injects the peroxide species into the RNR’s dimanga-nese active site. In one structure, a species resembling a peroxide is lodged inside this tunnel. “The binding site for this trapped species provides clues into how the pro-tein-protein complex might ensure safe

passage of a highly reactive oxidant to the active site,” Rosenzweig said.

The fact that this dimanganese RNR was so long assumed to be a diiron enzyme emphasizes that in vitro self-assembly of metalloproteins may not always reflect the proteins’ true composition inside cells, Stubbe noted. In earlier studies, scientists found that the enzyme would assemble in vitro with either diiron or dimanganese in the active site. But NrdI, required to acti-vate the dimanganese version, was always absent. As a consequence, scientists de-tected enzymatic activity only in the diiron version, and the RNR was branded a diiron enzyme.

Stubbe told C&EN that this is the only example she knows of in which a single protein can use two different metals and two different oxidants to do the same chemistry.

“Our results will open many scientists’ eyes to the complex and intriguing nature of metallocofactor biosynthesis in gen-eral,” Rosenzweig said. NrdI is just one example of the sophisticated biological machinery that organisms use to install and activate the appropriate metal cofac-tors in metalloproteins, she added.

Dimanganese RNRs might also point the way to new antibiotics. Human pathogens may have evolved these enzymes to outwit their host’s immune response, Bollinger suggested. Thus, the dimanganese RNR could present a useful new target for anti-bacterial design, he added. ■

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TWO FLAVORS E. coli’s dimanganese RNR enzyme can bind both Mn (purple, above) and Fe (green). Although the metals bind the same active-site amino acids, the metal coordination environments are distinct. The tyrosine where a radical must form to turn the enzymes on is shown in pink; red spheres denote water.

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