CORE Metadata, citation and similar papers at core.ac.uk Provided by Elsevier - Publisher Connector FEBS 29213 FEBS Letters 579 (2005) 745–748 The conserved histidine 106 of large thioredoxin reductases is likely to have a structural role but not a base catalyst function Judit Jacob, R. Heiner Schirmer, Stephan Gromer* Biochemiezentrum der Universita¨t Heidelberg (BZH), Im Neuenheimer Feld 504, D-69120 Heidelberg, Germany Received 26 November 2004; revised 3 January 2005; accepted 4 January 2005 Available online 11 January 2005 Edited by Hans Eklund of thioredoxin reductases exist: small TrxRs (subunit M ap- Abstract The catalytic activity of selenocysteine-containing r thioredoxin reductases can be mimicked by cysteine-variants if prox. 35 kDa) primarily present in bacteria, fungi, and plants the local environment at the C-terminal redox center supports and large TrxRs (subunit Mr approx. 55 kDa) present e.g. in thiol activation. This concept of a linear catalytic site was chal- higher eukaryotes. They differ in many aspects, such as the lenged by structural data suggesting that the invariant residue substrate spectrum, which is far broader for large than for His106 functions as a base catalyst for the dithiol-disulphide ex- small TrxRs [1]. change reaction between enzyme and substrate. As reported here, A key feature of the proposed mechanism of large thiore- 106 we changed His to asparagine, glutamine, and phenylalanine doxin reductases is the relatively flexible C-terminal tail which in various C-terminal mutants of Drosophila melanogaster thio- is responsible for the transport of electrons from the buried N- redoxin reductase. The catalytic activity dropped considerably, terminal redox-center near the flavin to bulky substrates at the yet pH-profiles did not reveal differences, rendering a function surface. This requires a second redox-active site located on the for His106 as a base catalyst unlikely. Interestingly, the phenyl- alanine-mutants, designed as negative controls were the most ac- C-terminal tail [3–5]. tive mutants which suggests rather a structural role of His106. The particular design of the second redox-center present in Ó 2005 Federation of European Biochemical Societies. Published large thioredoxin reductases differs between species: In mam- by Elsevier B.V. All rights reserved. malian TrxRs it is formed by a sequentially directly adjacent cysteine–selenocysteine-pair, whereas in the enzyme of Diptera Keywords: Thioredoxin reductase; Base catalyst; Mobile two cysteines form this motif [6,7]. Astonishingly, these cys- catalytic site; Thiol activation; Histidine functional analysis teine-variants are almost as active as their selenocysteine coun- terparts, despite the significantly higher pKa of cysteines and thus a reduced tendency to form the anions required for disul- 1. Introduction phide exchange reactions. Recent experiments strongly suggest that indeed sequentially neighbouring residues activate the thi- The thioredoxin system – formed by thioredoxin reductase ols [8]:InDrosophila melanogaster TrxR (containing the flexi- 1 (TrxR; EC 1.8.1.9 ), NADPH and its naming substrate thio- ble terminal sequence SCCS) hydroxyl groups of the flanking redoxin (Trx) – is an important constituent of the intracellular serines form hydrogen bonds with the thiols, lowering their redox milieu. It interacts with many different metabolic path- pKa and thus increase reactivity. ways such as DNA-synthesis, selenium metabolism and the It was, however, also suggested that a base catalyst facili- antioxidative network, and it exhibits significant species differ- tates the reaction between enzyme and substrate – with the ences. This renders the system an attractive and currently highly conserved His106, 2 located in close proximity to the extensively studied chemotherapeutic target in many fields of C-terminal Cys–Cys-pair [9], being the prime candidate (Fig. medicine – ranging from infectious diseases to cancer therapy 1A and B). This function of His106 was postulated in analogy (see Ref. [1] for review). to His464 which catalyzes the intramolecular dithiol-disulphide + TrxR (thioredoxin-S2 + NADPH + H thioredoxin- exchange between two cysteine pairs within the dimeric en- + (SH)2 + NADP ) is an NADPH-dependent flavoenzyme zyme (Fig. 1A and B, [10–13] and own observations, unpub- belonging to a family of homodimeric pyridine nucleotide- lished). disulphide oxidoreductases which includes enzymes like gluta- The objective of this work was to verify this hypothesis and thione reductase, lipoamide dehydrogenase, trypanothione thus provide further insight into the mechanistic details of reductase, and mercuric ion reductase [2]. Two distinct classes large thioredoxin reductases – a prerequisite for a rational, mechanism-based drug design. *Corresponding author. Fax: +49 6221 54 5586. E-mail address: [email protected] (S. Gromer). 2. Materials and methods 1 Thioredoxin reductase was formerly designated 1.6.4.5. Cloning, expression and purification of the 15 DmTrxR-1 mutants Abbreviations: Dm, Drosophila melanogaster; Sec, selenocysteine; and wild-type enzyme were performed applying established proce- (Dm)Trx, (Drosophila melanogaster) thioredoxin; (Dm)TrxR, (Dro- dures, as were enzyme kinetics (K , k ), pH-activity profiles, circular sophila melanogaster) thioredoxin reductase; -SCCS, -SCCG, -GCCS M cat and -GCCG, indicate the respective sequence of the C-terminal tetrapeptide of Dm TrxR in one letter code; DTNB, 5,50-dithio-bis- 2 The numbering of residues in the text refers to Drosophila melano- (2-nitrobenzoic acid) gaster TrxR-1 (AF301144). 0014-5793/$30.00 Ó 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2005.01.001 746 J. Jacob et al. / FEBS Letters 579 (2005) 745–748 0 Fig. 1. (A) Partial sequence alignment of large TrxRs and glutathione reductases. The conserved residues His106 (red) and His464 (blue) are highlighted. Alignments were done using the online-tool Clustal W 1.82 (www.ebi.ac.uk/clustalw). hTrxR-1 = human TrxR-1 (NCBI accession number: s66677), hTrxR-2 = human TrxR-2 (aad51324), SsTrxR = pig TrxR (aaf78791.1), mTrxR = mouse TrxR-1 (npÀ056577), CeTrxR = C. elegans TrxR (aad41826), AgTrxR = Anopheles gambiae TrxR (cad30858), DmTrxR-1 = D. melanogaster TrxR-1 (aag25640), hGR = human glutathione reductase 0 (rdhuu) (B) 3D-model of His106 (red) and His464 (blue) in D. melanogaster TrxR based on the structural information provided by protein data base (www.rcsb.org) file 1H6V of a Sec498 fi Cys rat TrxR mutant (subunits E and F) [9]. The C-terminal tail of one subunit is shown in green, with the adjacent cysteines highlighted in yellow. The second subunit, containing His106, is shown in dark gray. Also highlighted in yellow are the N-terminal cysteines and FAD (orange). The structure shown here was created using H. J. BernsteinÕs RasMol V 2.7.2.1.1 (www.bernstein-plus-sons.com/ software/rasmol). (C) Structural side-chain comparison of histidine and the amino acids used in the mutants of His106: glutamine, asparagine and phenylalanine. dichroism-analysis and absorbance spectra recordings. Details can be netic results (KM- and kcat-values differed by less than ±5%). found in this articleÕs online supplement, which is available at the Neither the absorbance-spectra (which showed typical flavo- FEBS-Letters homepage. The enzyme species are designated according protein characteristics) nor the circular dichroism spectra re- to their C-terminal tetrapeptide sequence (Gly–Cys–Cys–Gly = GCCG, Gly–Cys–Cys–Ser = GCCS, Ser–Cys–Cys–Gly = SCCG and vealed any significant differences between the mutants and Ser–Cys–Cys–Ser = SCCS = wild-type (wt)), and, furthermore, accord- wild-type enzyme, indicating an identical overall structure. If ing to the respective mutation at position 106. His106 indeed acted as an important base catalyst, one would expect a significant drop in activity for all three mutants, with the highest loss of activity for the phenylalanine mutant. This 3. Results and discussion was, however, not the case: Even though all three mutants were less active in thioredoxin reduction (kcat) than the His- The catalytic reactions on the Si-side of the flavin in large containing wild-type enzyme (Fig. 2A), the phenylalanine mu- thioredoxin reductases are essentially dithiol-disulphide ex- tants, originally designed as negative controls, were the most change reactions. In the course of these reactions a more reac- active ones (Fig. 2). The KM-values for thioredoxin remained tive intermediate, a thiolate or selenolate, has to be formed at essentially unaltered (Fig. 2A), indicating that the formation the C-terminal redox center of thioredoxin reductase [1,14]. of the Michaelis-complex is not affected. Moreover, no shift The question, why the catalytic activity of cysteine containing in the pH-profile (not shown, pH-optima at around 7.5–8.0) TrxRs, such as those from D. melanogaster or Anopheles gam- was observed for any of the mutants [8]. biae, almost matches that of their selenocysteine counterpart The exchange of the polar serines for glycines led to a signif- was addressed recently [8]. The hydroxyl groups of residues icant loss of activity. Thus these glycine-mutants should be sequentially adjacent to the redox-active C-terminal Cys– even more sensitive to an exchange of His106 for Asn, Gln or 106 Cys-pair seem to lower the thiols’ pKa, facilitating thiolate even Phe if histidine acted as a base catalyst. Yet, this was formation. not the case either. We changed