Kurth Julia M, Butt Julea N, Kelly David J, Dahl Christiane
Institut für Mikrobiologie & Biotechnologie, Rheinische Friedrich-Wilhelms-Universität Bonn, D-53115 Bonn, Germany.
Centre for Molecular and Structural Biochemistry, School of Chemistry, and School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, U.K.
Biosci Rep. 2016 Dec 9;36(6). doi: 10.1042/BSR20160457. Print 2016 Dec.
Bifunctional dihaem cytochrome c thiosulfate dehydrogenases/tetrathionate reductases (TsdA) exhibit different catalytic properties depending on the source organism. In the human food-borne intestinal pathogen Campylobacter jejuni, TsdA functions as a tetrathionate reductase enabling respiration with tetrathionate as an alternative electron acceptor. In the present study, evidence is provided that Cys and Met serve as the sixth ligands of Haem 1 and Haem 2 respectively, in the oxidized CjTsdA wt protein. Replacement of Cys resulted in a virtually inactive enzyme, confirming Haem 1 as the active site haem. Significantly, TsdA variants carrying amino acid exchanges in the vicinity of the electron-transferring Haem 2 (Met, Asn and Lys) exhibited markedly altered catalytic properties of the enzyme, showing these residues play a key role in the physiological function of TsdA. The growth phenotypes and tetrathionate reductase activities of a series of ΔtsdA/*tsdA complementation strains constructed in the original host C. jejuni 81116, showed that in vivo, the TsdA variants exhibited the same catalytic properties as the pure, recombinantly produced enzymes. However, variants that catalysed tetrathionate reduction more effectively than the wild-type enzyme did not allow better growth.
双功能二血红素细胞色素c硫代硫酸盐脱氢酶/连四硫酸盐还原酶（TsdA）根据来源生物体的不同表现出不同的催化特性。在食源性人类肠道病原体空肠弯曲菌中，TsdA作为连四硫酸盐还原酶发挥作用，使细胞能够以连四硫酸盐作为替代电子受体进行呼吸作用。在本研究中，有证据表明，在氧化态的空肠弯曲菌野生型TsdA（CjTsdA wt）蛋白中，半胱氨酸（Cys）和甲硫氨酸（Met）分别作为血红素1（Haem 1）和血红素2（Haem 2）的第六个配体。半胱氨酸的替换导致酶几乎失活，证实血红素1为活性位点血红素。值得注意的是，在电子传递血红素2附近携带氨基酸替换（甲硫氨酸、天冬酰胺和赖氨酸）的TsdA变体表现出明显改变的酶催化特性，表明这些残基在TsdA的生理功能中起关键作用。在原始宿主空肠弯曲菌81116中构建的一系列ΔtsdA/*tsdA互补菌株的生长表型和连四硫酸盐还原酶活性表明，在体内，TsdA变体表现出与纯的重组产生的酶相同的催化特性。然而，比野生型酶更有效地催化连四硫酸盐还原的变体并不能使细菌更好地生长。
