Proc. Natl. Acad. Sci. USA Vol. 94, pp. 3621–3626, April 1997 Biochemistry The mechanism of thioredoxin reductase from human placenta is similar to the mechanisms of lipoamide dehydrogenase and glutathione reductase and is distinct from the mechanism of thioredoxin reductase from Escherichia coli L. DAVID ARSCOTT*, STEPHAN GROMER†,R.HEINER SCHIRMER†,KATJA BECKER†, AND CHARLES H. WILLIAMS,JR.*‡§ *Department of Veterans Affairs Medical Center and ‡Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48105; and †Institute of Biochemistry II, Heidelberg University, 69120 Heidelberg, Germany Communicated by Vincent Massey, University of Michigan School of Medicine, Ann Arbor, MI, January 31, 1997 (received for review December 23, 1996) ABSTRACT Thioredoxin reductase, lipoamide dehydro- archaeon Methanococcus jannaschii (1–2). The three- genase, and glutathione reductase are members of the pyri- dimensional structure of TrxR from Escherichia coli (eTrxR) dine nucleotide–disulfide oxidoreductase family of dimeric is known, and its mechanism has been studied extensively flavoenzymes. The mechanisms and structures of lipoamide (3–4). On the basis of significant amino acid sequence simi- dehydrogenase and glutathione reductase are alike irrespec- larities and the presence of a redox-active disulfide and the tive of the source (subunit Mr '55,000). Although the mech- substrate specificities (pyridine nucleotides and disulfide com- anism and structure of thioredoxin reductase from Esche- pounds), eTrxR has been placed in the pyridine nucleotide– richia coli are distinct (Mr '35,000), this enzyme must be disulfide oxidoreductase family that includes lipoamide dehy- placed in the same family because there are significant amino drogenase (LipDH) and glutathione reductase (GR) (5–6). acid sequence similarities with the other two enzymes, the The three-dimensional structures of the latter enzymes are presence of a redox-active disulfide, and the substrate speci- known (7–9). Their mechanisms have been studied extensively ficities. Thioredoxin reductase from higher eukaryotes on the and have been found to be very similar to each other over a other hand has a M of 55,000 [Luthman, M. & Holmgren, r ' wide range of organisms. Their structures and mechanisms are A. (1982) Biochemistry 21, 6628–6633; Gasdaska, P. Y., Gas- rather different from those of eTrxR. In particular, the subunit daska, J. R., Cochran, S. & Powis, G. (1995) FEBS Lett 373, M are 35,000 for eTrxR and 55,000 for LipDH and GR (6). 5–9; Gladyshev, V. N., Jeang, K. T. & Stadtman, T.C. (1996) r Proc. Natl. Acad. Sci. USA 93, 6146–6151]. Thus, the evolution TrxR isolated from various mammalian tissues were found of this family is highly unusual. The mechanism of thioredoxin to have a subunit Mr of '55,000 (10). Moreover, complete reductase from higher eukaryotes is not known. As reported DNA sequences have been published for TrxR from humans here, thioredoxin reductase from human placenta reacts with (1, 11) and Plasmodium falciparum (ref. 12 and references only a single molecule of NADPH, which leads to a stable therein). The deduced amino acid sequences of these high Mr intermediate similar to that observed in titrations of lipoam- TrxR are much more closely related to the sequences of GR ide dehydrogenase or glutathione reductase. Titration of from humans (35% identity) or E. coli (40% identity) than to thioredoxin reductase from human placenta with dithionite the sequence of eTrxR (24% identity) (1, 13). Low Mr TrxR takes place in two spectral phases: formation of a thiolate– have been found not only in E. coli but also in the anaerobe flavin charge transfer complex followed by reduction of the Eubacterium acidoaminophilum, the archaeon Methanococcus flavin, just as with lipoamide dehydrogenase or glutathione jannaschii, and the facultative phototroph Rhodobacter spha- reductase. The first phase requires more than one equivalent eroides Y, as well as a number of fungi and plants (1, 2, 14–17, of dithionite. This suggests that the penultimate seleno- and references in ref. 1). In addition, a TrxR-like enzyme has cysteine [Tamura, T. & Stadtman, T.C. (1996) Proc. Natl. been isolated from Salmonella typhimurium that, together with Acad. Sci. USA 93, 1006–1011] is in redox communication with AhpC, acts as an alkyl hydroperoxide reductase. AhpC is a the active site disulfideydithiol. Nitrosoureas of the carmus- 22-kDa protein that, like Trx, has a redox-active disulfidey tine type inhibit only the NADPH reduced form of human dithiol (18, 19). A reductase having similar activity has been thioredoxin reductase. These compounds are widely used as isolated from Amphibacillus xylanus (20). These reductases are cytostatic agents, so this enzyme should be studied as a target homologous with low Mr TrxR (19). On the basis of the in cancer chemotherapy. In conclusion, three lines of evidence presently available database, high and low Mr TrxR are mu- indicate that the mechanism of human thioredoxin reductase tually exclusive in the same organism. It is, however, premature is like the mechanisms of lipoamide dehydrogenase and glutathione reductase and differs fundamentally from the mechanism of E. coli thioredoxin reductase. Abbreviations: TrxR, thioredoxin reductases; BCNU, 1,3-bis(2- chloroethyl)-1-nitrosourea; EH2, 2-electron reduced enzyme, primar- ily thiolate-flavin charge transfer complex, EH4, 4-electron reduced Thioredoxin reductases (TrxR) are homodimeric flavoen- enzyme with reduced FAD and an active site dithiol; Eox, oxidized zymes occurring, or implied by gene sequence to occur, in a enzyme containing an active site disulfide; eq, equivalents relative to enzyme bound FAD; eTrxR, TrxR from E. coli; GR, glutathione wide variety of organisms—prokaryotes, eukaryotes, and the reductase; hTrxR, TrxR from human placenta; LipDH, lipoamide dehydrogenase; SeCys, selenocysteine. It should be noted that oxi- The publication costs of this article were defrayed in part by page charge dized enzyme (Eox), 2-electron reduced enzyme (EH2), and 4-electron payment. This article must therefore be hereby marked ‘‘advertisement’’ in reduced enzyme (EH4) refer only to the Cys pair/FAD-ensemble at the accordance with 18 U.S.C. §1734 solely to indicate this fact. catalytic site. Other titratable redox centers of the enzyme are not included in these definitions. Copyright q 1997 by THE NATIONAL ACADEMY OF SCIENCES OF THE USA §To whom reprint requests should be addressed at: Medical Research 0027-8424y97y943621-6$2.00y0 Service, 151, VA Medical Center, 2215 Fuller Road, Ann Arbor, MI PNAS is available online at http:yywww.pnas.org. 48105. 3621 Downloaded by guest on September 28, 2021 3622 Biochemistry: Arscott et al. Proc. Natl. Acad. Sci. USA 94 (1997) MATERIALS AND METHODS TrxR from Human Placenta (hTrxR). hTrxR was purified according to Oblong et al. (23) with modifications (S.G., K.B., and H.S., unpublished work). As a final step, the enzyme was eluted from a 2959-ADP–Sepharose column using 0.7 mM NADP1 in 50 mM TriszHCly1 mM EDTA buffer of pH 7.6; NADP1 was subsequently removed by washing with this buffer without NADP1 in a Centriprep 30 (Amicon). Isolated TrxR ('1.0 mg from one placenta of 550 g) had a specific activity of 35 unitsymg when tested in the 5,59- dithiobis(2-nitrobenzoate) reduction assay (23, 28). Enzyme concentration was estimated by measuring the A463 nm and applying an assumed extinction coefficient of 11.3 mM21zcm21 per subunit of Eox. As revealed by SDSyPAGE analysis, the preparation was .95% pure and had—as an apoprotein—an apparent subunit Mr of '57,000, which corresponds to a value of 58,000 for the FAD-containing subunit. Disulfide reducta- ses tend to exhibit a 5–10% higher Mr in SDSyPAGE analyses than in amino acid sequence analyses, so we recommend using a Mr value of 55,000 (1, 11). The selenium content as determined by atomic absorption FIG. 1. Phylogenetic tree. The distances between branch points are spectroscopy was 0.93 6 0.02 molymol subunit. 0.98 6 0.06 mol not intended to reflect time quantitatively. A gene sequence from the FADymol TrxR subunit was found using the apoGR comple- worm (Caenorhabditis elegans) is homologous with the sequence of mentation test (29). An enzyme of 35 unitsymg was used in the hTrxR (1). dithionite titration experiment (see below). A slightly less pure preparation (32 unitsymg) was used to measure the presteady to assign the two different forms of TrxR to individual state kinetics (see below). branching points of the phylogenetic tree. Equilibrium Titration with Dithionite. Approximately 17 In any case, the evolution of TrxR has followed at least two mM of enzyme in 1 ml of 50 mM potassium phosphatey2mM courses (Fig. 1). One pathway can be explained by the con- EDTA, pH 7.4, containing 0.1 equivalents relative to enzyme- tinuous Apollonian divergent evolution of the ancestor of bound FAD (eq) methyl viologen as a carrier of reducing LipDH, GR, and high Mr TrxR. In contrast, for the develop- equivalents was degassed by eight cycles of vacuum and pure ment of low Mr TrxR, the same modules, i.e., the NADPH- nitrogen gas and titrated with a solution of anaerobic dithio- binding and the FAD-binding domains, as in the high Mr nite, which had been standardized by titrating lumiflavin-3- counterpart are used, but the active site and subunit interface acetic acid (30). Spectra were recorded at 258C using a exhibit a completely different design. In addition, the catalytic Perkin–Elmer spectrophotometer. At 3 points in the first activity is accompanied by domain movements in low Mr TrxR phase, the titration was suspended to allow thorough equili- (3). Thus, the evolution of the TrxR is characterized by radical bration of reducing equivalents between all redox-active com- Dionysian divergence from an ancestral structure followed by ponents of the enzyme. convergent evolution to a common function (Fig. 1).