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A Three-dimensional Picture of Methyl Transfer

Several enzymes can synthesize methionine by methylating the thiol group of homocysteine. One of these enzymes, cobalamin-independent methionine synthase (MetE), catalyzes the direct transfer of the methyl group of N⁵-methyltetrahydrofolate (CH₃-H₄PteGlun) to the sulfur atom of homocysteine. Jean-Luc Ferrer and colleagues now provide significant insight into the mechanism of action of this methyl transfer based on the crystal structure of MetE from Arabidopsis thaliana.

Cobalamin-independent methionine synthase contains an active site cleft which binds methionine and PteGlu5.

The investigators solved the structure of MetE free and complexed with homocysteine or folate substrates. They found that MetE is a monomer consisting of two ()₈ barrel domains with a deep groove at their interface. The active site of the enzyme is located at the surface of the C-terminal domain, facing the large interdomain cleft. Inside the active site cleft, several residues on the C-terminal domain are involved in interactions with a zinc ion, homocysteine/methionine, and CH₃-H₄PteGlu_n. Ferrer et al. found that their structures are not consistent with a proposed model for methyl transfer in which homocysteine is activated by direct coordination with zinc, causing it to become a strong nucleophile that attacks CH₃-H₄PteGlu_n. The crystal structures show that homocysteine binding does not lead to direct coordination of the sulfur atom to zinc, leaving the door open for a new model for methyl transfer.

FOOTNOTES

See referenced article, J. Biol. Chem. 2004, 279, 44235–44238

Probes Shed Light on DNA Handoff

Because DNA is constantly exposed to an onslaught of mutagens, cells must have a method for excising and repairing DNA lesions. Nucleotide excision repair (NER) is a major DNA repair pathway involving the UvrABC proteins. In bacteria, UvrA dimerizes to become UvrA₂, which then interacts with UvrB to form the UvrA₂B complex. The complex then binds to DNA and searches for lesions via the -hairpin of UvrB. When a lesion is encountered, conformational changes occur, and the DNA, which is initially in contact with UvrA, is passed to UvrB via an unknown mechanism. Once this happens, UvrC binds to the UvrB·DNA complex and excises the lesion.

Putative UvrB DNA binding model showing potential points of contact for cross-linking reagents.

To capture UvrA in the act of DNA handoff to UvrB, Matthew J. DellaVecchia and colleagues designed two types of arylazido-modified photoaffinity cross-linking reagents. One type of reagent probed Uvr residues closest to the damaged DNA strand, and one probed residues closest to the non-damaged strand. The damaged strand probes, which functioned as both lesions and cross-linkers, consisted of dNTP analogues linked to terminal arylazido groups. The non-damaged strand probe consisted of an arylazido compound coupled to a phosphorothioate-modified backbone of an oligonucleotide opposite the damaged strand, which contained an internal fluorescein adduct. Using their probes, DellaVecchia et al. determined that DNA handoff occurs in three steps: 1) UvrA and UvrB bind to DNA via contact sites on UvrA; 2) a transfer reaction occurs with UvrB mostly contacting the non-damaged strand; 3) the lesion is engaged by the damage recognition pocket of UvrB, and UvrA is released. The methods developed here should be applicable to other DNA enzymes as well and are likely to be very useful to all DNA enzymologists.

FOOTNOTES

See referenced article, J. Biol. Chem. 2004, 279, 45245–45256