tailieunhanh - Báo cáo khoa học: A single intersubunit salt bridge affects oligomerization and catalytic activity in a bacterial quinone reductase

YhdA, a thermostable NADPH:FMN oxidoreductase fromBacillus subtil-is, reduces quinones via a ping-pong bi-bi mechanism with a pronounced preference for NADPH. The enzyme occurs as a stable tetramer in solu-tion. The two extended dimer surfaces are packed against each other by a 90 rotation of one dimer with respect to the other. | ỊFEBS Journal A single intersubunit salt bridge affects oligomerization and catalytic activity in a bacterial quinone reductase Alexandra Binter1 Nicole Staunig2 Ilian Jelesarov3 Karl Lohner4 Bruce A. Palfey5 Sigrid Deller1 Karl Gruber2 and Peter Macheroux1 1 Institute of Biochemistry Graz University of Technology Austria 2 Institute of Molecular Biosciences University of Graz Austria 3 Institute of Biochemistry University of Zurich Switzerland 4 Institute of Biophysics and Nanosystems Research Austrian Academy of Sciences Graz Austria 5 Department of BiologicalChemistry University of Michigan Ann Arbor MI USA Keywords NADPH FMN oxidoreductase oligomerization quinone reductase salt bridge thermostability Correspondence K. Gruber Institute of Molecular Biosciences University of Graz Humboldtstrasse 50 III A-8010 Graz Austria Fax 43 316 380 9897 Tel 43 316 380 5483 E-mail P. Macheroux Institute of Biochemistry Graz University of Technology Petersgasse 12 II A-8010 Graz Austria Fax 43 316 873 6952 Tel 43 316 873 6450 E-mail Received 25 May 2009 revised 14 July 2009 accepted 17 July 2009 doi YhdA a thermostable NADPH FMN oxidoreductase from Bacillus subtil-is reduces quinones via a ping-pong bi-bi mechanism with a pronounced preference for NADPH. The enzyme occurs as a stable tetramer in solution. The two extended dimer surfaces are packed against each other by a 90 rotation of one dimer with respect to the other. This assembly is stabilized by the formation of four salt bridges between K109 and D137 of the neighbouring protomers. To investigate the importance of the ion pair contacts the K109L and D137L single replacement variants as well as the K109L D137L and K109D D137K double replacement variants were generated expressed purified crystallized and biochemically characterized. The K109L and D137L variants form dimers instead of tetramers whereas the K109L D137L and K109D D137K .