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Flavocytochrome C - a novel fumarate reductase from Shewanella putrefaciens
HAEM IRON
F022
311
F L A V O C Y T O C H R O M E C - A N O V E L FUMARATE R E D U C T A S E F R O M S H E W A N E L L A PUTREFACIENS.
S.L. Pealing, A.C. Black a, S.K. Chapman b, F.B. Ward a, G~A. Reid a.
alnstitute of Cell and Molecular Biology and bDepartment of Chemistry, University of Edinburgh, King's Buildings, Edinburgh, U.K. Flavocytochrome c is a soluble enzyme located in the periplasmic space of Shewanella putrefaciens. It has a single subunit of 84 kDa as estimated by SDS-PAGE and behaves as a monomer under non-denaturing conditions. Sequence from DNA encoding the N-terminal portion of the polypeptide shows that the protein is apparantly synthesised with a typical signal sequence responsible for targeting the protein to the periplasm. Cleavage between Ala25 and Ala26 results in release of the mature polypeptide.The N-terminal domain of about 100 amino acids contains four typical haem-attachment sites (CXXCH) but otherwise shows no significant sequence similarity to other multi-haem cytochromes. The "haem domain" is followed by a putative flavin domain which contains a typical FAD binding region. This part of the sequence shows considerable similarity to mercuric reductases, lipoamide dehydrogenases and other flavoenzymes. However, flavocytochrome c is very different from the well characterized fumarate reductase of Escherichia coli which is membrane-bound and contains four types of subunit, none of which is a cytochrome [1]. The expression of S. putrefaciens tlavocytochrome c is specifically induced by anaerobiosis suggesting that the enzyme functions in anaerobic transfer pathways [2]. A role for the flavocytochrome c as a fumarate reductase is supported by zymogram staining and by the fact that the purified enzyme has a high affinity for fumarate (K m = 15 uM) and efficiently catalyzes electron transfer to fumarate from the artificial donor methyl viologen (kcat = 278s-1). The enzyme is essentially a unidirectional fumarate reductase since succinate dehydrogenase activity is very low (kcat -- 0.05 s-1, K m = 350 uM). This is in marked contrast to the membrane bound fumarate reductases where forward and reverse reactions are efficiently catalysed.
Io
2.
P. Dickie and H. Weiner, Can JBiochern., 57, 813 (1979). C.I. Morris, D.M. Gibson and F.B. Ward, FEMS Microbiol. Lett., 69, 259 (1900).
F022
311
F L A V O C Y T O C H R O M E C - A N O V E L FUMARATE R E D U C T A S E F R O M S H E W A N E L L A PUTREFACIENS.
S.L. Pealing, A.C. Black a, S.K. Chapman b, F.B. Ward a, G~A. Reid a.
alnstitute of Cell and Molecular Biology and bDepartment of Chemistry, University of Edinburgh, King's Buildings, Edinburgh, U.K. Flavocytochrome c is a soluble enzyme located in the periplasmic space of Shewanella putrefaciens. It has a single subunit of 84 kDa as estimated by SDS-PAGE and behaves as a monomer under non-denaturing conditions. Sequence from DNA encoding the N-terminal portion of the polypeptide shows that the protein is apparantly synthesised with a typical signal sequence responsible for targeting the protein to the periplasm. Cleavage between Ala25 and Ala26 results in release of the mature polypeptide.The N-terminal domain of about 100 amino acids contains four typical haem-attachment sites (CXXCH) but otherwise shows no significant sequence similarity to other multi-haem cytochromes. The "haem domain" is followed by a putative flavin domain which contains a typical FAD binding region. This part of the sequence shows considerable similarity to mercuric reductases, lipoamide dehydrogenases and other flavoenzymes. However, flavocytochrome c is very different from the well characterized fumarate reductase of Escherichia coli which is membrane-bound and contains four types of subunit, none of which is a cytochrome [1]. The expression of S. putrefaciens tlavocytochrome c is specifically induced by anaerobiosis suggesting that the enzyme functions in anaerobic transfer pathways [2]. A role for the flavocytochrome c as a fumarate reductase is supported by zymogram staining and by the fact that the purified enzyme has a high affinity for fumarate (K m = 15 uM) and efficiently catalyzes electron transfer to fumarate from the artificial donor methyl viologen (kcat = 278s-1). The enzyme is essentially a unidirectional fumarate reductase since succinate dehydrogenase activity is very low (kcat -- 0.05 s-1, K m = 350 uM). This is in marked contrast to the membrane bound fumarate reductases where forward and reverse reactions are efficiently catalysed.
Io
2.
P. Dickie and H. Weiner, Can JBiochern., 57, 813 (1979). C.I. Morris, D.M. Gibson and F.B. Ward, FEMS Microbiol. Lett., 69, 259 (1900).