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Schematic Representation of the Reaction Mechanism of N. gonorrhoeae MsrBNumbering of amino acids is based on the mouse MsrB1 sequence. A water molecule is indicated as W. This reaction mechanism was adapted from Lowther et al. [20]. The nucleophilic attack by Cys95 on sulfoxide moiety of the substrate results in a trigonal-bipyramidal intermediate (Ia), followed by the formation of the sulfenic acid intermediate of Cys95 and the release of methionine (Ib). The resolving Cys41 attacks the sulfenic acid intermediate of Cys95 to form a disulfide bond (II). The disulfide bond is then reduced by Trx in vivo or by DTT in vitro and the active site is returned to the fully reduced state (III).
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pbio-0030375-g002: Schematic Representation of the Reaction Mechanism of N. gonorrhoeae MsrBNumbering of amino acids is based on the mouse MsrB1 sequence. A water molecule is indicated as W. This reaction mechanism was adapted from Lowther et al. [20]. The nucleophilic attack by Cys95 on sulfoxide moiety of the substrate results in a trigonal-bipyramidal intermediate (Ia), followed by the formation of the sulfenic acid intermediate of Cys95 and the release of methionine (Ib). The resolving Cys41 attacks the sulfenic acid intermediate of Cys95 to form a disulfide bond (II). The disulfide bond is then reduced by Trx in vivo or by DTT in vitro and the active site is returned to the fully reduced state (III).

Mentions: A crystal structure of an MsrB domain of Neisseria gonorrhoeae PilB has been reported [20]. This domain corresponds to a Cys-containing MsrB. pKa values of Cys thiols are typically around 8.3, unless adjusted by microenvironmental conditions, whereas the selenol group of Sec is fully ionized at physiological pH owing to its low pKa value of 5.2 [4,5]. The N. gonorrhoeae MsrB structure suggested that the catalytic Cys95 nucleophile is activated by a Cys95–Arg93–Asp85 triad [20] (Figure 1B), which is conserved in all MsrBs except for replacement of Asp with Glu in some homologs. It was also proposed, based on the N. gonorrhoeae MsrB structure, that His77 and Asn97 form hydrogen bonds with a water molecule, which in turn interacts with the oxygen atom of the sulfoxide moiety of the substrate [20] (Figure 2). This hydrogen-bond network may stabilize the intermediate in the reaction [20]. Thus, the residues conserved in the Cys-containing MsrBs, but absent in selenoprotein MsrBs (see Figure 1), are part of the active site.

Different Catalytic Mechanisms in Mammalian Selenocysteine- and Cysteine-Containing Methionine-R-Sulfoxide ReductasesSelenoproteins-Tracing the Role of a Trace Element in Protein FunctionSelenium Speeds Reactions

Kim HY, Gladyshev VN - PLoS Biol. (2005)

Bottom Line: Selenocysteine (Sec) is found in active sites of several oxidoreductases in which this residue is essential for catalytic activity.We prepared Sec-containing forms of MsrB2 and MsrB3 and found that they were more than 100-fold more active than the natural Cys forms.The data also suggested that Sec- and Cys-containing oxidoreductases require distinct sets of active-site features that maximize their catalytic efficiencies and provide strategies for protein design with improved catalytic properties.

Affiliation: Department of Biochemistry, University of Nebraska, Lincoln, Nebraska, USA.

ABSTRACT
Selenocysteine (Sec) is found in active sites of several oxidoreductases in which this residue is essential for catalytic activity. However, many selenoproteins have fully functional orthologs, wherein cysteine (Cys) occupies the position of Sec. The reason why some enzymes evolve into selenoproteins if the Cys versions may be sufficient is not understood. Among three mammalian methionine-R-sulfoxide reductases (MsrBs), MsrB1 is a Sec-containing protein, whereas MsrB2 and MsrB3 contain Cys in the active site, making these enzymes an excellent system for addressing the question of why Sec is used in biological systems. In this study, we found that residues, which are uniquely conserved in Cys-containing MsrBs and which are critical for enzyme activity in MsrB2 and MsrB3, were not required for MsrB1, but increased the activity of its Cys mutant. Conversely, selenoprotein MsrB1 had a unique resolving Cys reversibly engaged in the selenenylsulfide bond. However, this Cys was not necessary for activities of either MsrB2, MsrB3, or the Cys mutant of MsrB1. We prepared Sec-containing forms of MsrB2 and MsrB3 and found that they were more than 100-fold more active than the natural Cys forms. However, these selenoproteins could not be reduced by the physiological electron donor, thioredoxin. Yet, insertion of the resolving Cys, which was conserved in MsrB1, into the selenoprotein form of MsrB3 restored the thioredoxin-dependent activity of this enzyme. These data revealed differences in catalytic mechanisms between selenoprotein MsrB1 and non-selenoproteins MsrB2 and MsrB3, and identified catalytic advantages and disadvantages of Sec- and Cys-containing proteins. The data also suggested that Sec- and Cys-containing oxidoreductases require distinct sets of active-site features that maximize their catalytic efficiencies and provide strategies for protein design with improved catalytic properties.

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