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Hpy188I-DNA structures – snapshots of the GIY-YIG nuclease mediated catalysis

2011; Wiley; Volume: 67; Issue: a1 Linguagem: Inglês

10.1107/s0108767311080159

1600-5724

H. Czapinska, M. Sokolowska, Matthias Bochtler,

Advanced biosensing and bioanalysis techniques

SessionsC784 signifi cantly modify the structural properties and organization of the protein.The crystal structure of TmPrxNtrC40S has been determined for the oxidized form and for the reduced form in presence of hydrogen sulfi te, as well as two crystalline forms of the TmPrx module.The full-length protein TmPrxNtr, which contains a FMN prosthetic group, displays nitroreductase, peroxidase and quinone, fl avine, chromate and iron reductase activities using either NADH or NADPH as a donor.The mutation of the catalytic cysteine of the Prx module (TmPrxNtrC40S) does not alter the catalytic effi ciency of the protein suggesting that the activity measured does not require the Prx module.Both modules TmPrx and TmNtr have peroxidase activity but the oxidized enzyme is not regenerated in the same way.NAD(P)H is the direct reductant for TmNtr while TmPrx is regenerated in vitro via glutaredoxins or thioredoxins.In addition TmPrx is able to reduce H 2 O 2 and COOH whereas TMPrxNtr only reduces H 2 O 2 .The TmPrxNtrC40S enzyme crystallizes as a dimer, the active form of the protein.The Ntr module of one monomer is in contact with the Ntr and Prx modules of the other one but no contact is observed between the two Prx modules.On the contrary, in the crystalline forms of the isolated domain, two TmPrx molecules are linked by an intermolecular disulfi de bond.In TmPrxNtrC40S, the FMN prosthetic groups bind in deep pockets at the dimer interface and interact with elements of both monomers.An ion sulphate is positioned by hydrogen bonding to the catalytic site of the Prx modules.In the reduced TmPrxNtrC40S crystal, one sulphate is replaced by a bisulfi te introducing structural modifi cations to the associated Prx module.Altogether, the results indicate that the TmPrx and TmNtr modules apparently function independently, each possessing its own peroxidase activity, with the TmNtr module having diaphorase activity linked to the FMN moiety versus a broad range of substrates.This raises the question of their fusion which is specifi cally found in thermotogales.