Crystal Structure and Catalysis of the Selenoprotein Thioredoxin Reductase 1

Qing Cheng,Tatyana Sandalova,Ylva Lindqvist,Elias S.J Arnér

doi:10.1074/jbc.m807068200

Abstract

Selenoproteins contain a highly reactive 21st amino acid selenocysteine (Sec) encoded by recoding of a predefined UGA codon. Because of a lack of selenoprotein supply, high chemical reactivity of Sec, and intricate translation machineries, selenoprotein crystal structures are difficult to obtain. Structural prerequisites for Sec involvement in enzyme catalysis are therefore sparsely known. Here we present the crystal structure of catalytically active rat thioredoxin reductase 1 (TrxR1), revealing surprises at the C-terminal Sec-containing active site in view of previous literature. The oxidized enzyme presents a selenenylsulfide motif in trans-configuration, with the selenium atom of Sec-498 positioned beneath the side chain of Tyr-116, thereby located far from the redox active moieties proposed to be involved in electron transport to the Sec-containing active site. Upon reduction to a selenolthiol motif, the Sec residue moved toward solvent exposure, consistent with its presumed role in reduction of TrxR1 substrates or as target of electrophilic agents inhibiting the enzyme. A Y116I mutation lowered catalytic efficiency in reduction of thioredoxin, but surprisingly increased turnover using 5-hydroxy-1,4-naphthoquinone (juglone) as substrate. The same mutation also decreased sensitivity to inhibition by cisplatin. The results suggest that Tyr-116 plays an important role for catalysis of TrxR1 by interacting with the selenenylsulfide of oxidized TrxR1, thereby facilitating its reduction in the reductive half-reaction of the enzyme. The interaction of a selenenylsulfide with the phenyl ring of a tyrosine, affecting turnover, switch of substrate specificity, and modulation of sensitivity to electrophilic agents, gives important clues into the mechanism of TrxR1, which is a selenoprotein that plays a major role for mammalian cell fate and function. The results also demonstrate that a recombinant selenoprotein TrxR can be produced in high amount and sufficient purity to enable crystal structure determination, which suggests that additional structural studies of these types of proteins are feasible.

Highlights

thioredoxin reductase 1 (TrxR1) Crystallization, Data Collection, and Refinement— Numerous attempts to crystallize wild type TrxR1 in a wide range of crystallization conditions were performed, using more than 200 mg of TrxR1 having a specific activity of at least 40 units/mg, which is a slightly higher enzymatic activity than that of native TrxR1 purified from calf liver and thymus [23] or rat liver [24]
The Sec-containing wild type TrxR1 crystals were obtained at conditions different from Sec-498 to Cys mutant rat protein made previously [32], they belonged to the same space group P21 [32] and had similar, but not identical, cell dimensions
We modeled the TrxR1-Trx intermediary complex as a mixed selenenylsulfide between Cys-32 of Trx and Sec-498 of TrxR1, which could readily be built with only very minor shifts in the active site cleft upon regularization (Fig. 3, A and B)

Summary

Introduction

It has not been clear, what intermediates are formed during this reduction and what residues are the leaving groups. The location of the selenenylsulfide motif as observed in the structure would not be compatible with a direct interaction with the FAD/dithiol motif. One possibility would be a reduction of the selenenylsulfide through an electron relay through several additional residues from the N-terminal domain, even through a potential Tyr-116 tyrosyl radical intermediate, thereby participating in a long range selenenylsulfide reduction. We still find a direct interchange between the C-terminal disulfide and the N-terminal dithiol motifs to be the most probahydroxyl group, interacted with the selenenylsulfide of the ble event, which, necessitates that the selenenylsul-

Results

Discussion

Conclusion