Rhodobacter sphaeroides methionine sulfoxide reductase P reduces R- and S-diastereomers of methionine sulfoxide from a broad-spectrum of protein substrates

Abstract

Methionine (Met) is prone to oxidation and can be converted to Met sulfoxide (MetO), which exists as R- and S-diastereomers. MetO can be reduced back to Met by the ubiquitous methionine sulfoxide reductase (Msr) enzymes. Canonical MsrA and MsrB were shown to be absolutely stereospecific for the reduction of S-diastereomer and R-diastereomer, respectively. Recently, a new enzymatic system, MsrQ/MsrP which is conserved in all gram-negative bacteria, was identified as a key actor for the reduction of oxidized periplasmic proteins. The haem-binding membrane protein MsrQ transmits reducing power from the electron transport chains to the molybdoenzyme MsrP, which acts as a protein-MetO reductase. The MsrQ/MsrP function was well established genetically, but the identity and biochemical properties of MsrP substrates remain unknown. In this work, using the purified MsrP enzyme from the photosynthetic bacteria Rhodobacter sphaeroides as a model, we show that it can reduce a broad spectrum of protein substrates. The most efficiently reduced MetO is found in clusters, in amino acid sequences devoid of threonine and proline on the C-terminal side. Moreover, R. sphaeroides MsrP lacks stereospecificity as it can reduce both R- and S-diastereomers of MetO, similarly to its Escherichia coli homolog, and preferentially acts on unfolded oxidized proteins. Overall, these results provide important insights into the function of a bacterial envelop protecting system, which should help understand how bacteria cope in harmful environments.

Susceptibility of Protein Methionine Oxidation in Response to Hydrogen Peroxide Treatment–Ex Vivo Versus In Vitro: A Computational Insight

“…This seems to be the case at least for MsrP in Rhodobacter sphaeroides . Indeed, Tarrago and co-workers have shown that when the periplasmic extracts of this bacterium are treated with NaOCl and then incubated with MsrP, those MetO sites next to glutamate and/or aspartate were more efficiently reduced back to methionine [ 9 ].…”

“…Canonical MsrA and MsrB have been shown to be absolutely stereospecific for the reduction of Met-S-O and Met-R-O, respectively [ 8 ]. More recently, a new enzymatic system, MsrQ/MsrP, which can reduce both R- and S-diasteroisomers has been described in gram-negative bacteria [ 9 ]. Thus, like phosphorylation, methionine oxidation is a reversible covalent modification that has been demonstrated to both up regulate [ 10 , 11 , 12 ] and down regulate [ 13 , 14 ] protein activity through the direct sulfoxidation of specific methionine residues.…”

Reduction of Protein Bound Methionine Sulfoxide by a Periplasmic Dimethyl Sulfoxide Reductase

“…By contrast, the enzymes belonging to the DMSO reductase family have a more complex catalytic center constituted of two molybdopterin guanosine dinucleotides coordinating the Mo atom [18]. The periplasmic methionine sulfoxide reductase (MsrP) belongs to the sulfite oxidase family and was recently shown to be a non-stereospecific protein-MetO reductase with a broad range of protein substrates [19,20]. MsrP is present in most Gram-negative bacteria and genetic characterizations in Escherichia coli and Azospira suillum indicated that it is principally involved in protein oxidation protection during stress induced by reactive chlorine species [19,21].…”

“…The MsrA, MsrB, and MsrP are the only enzymes for which the ability to reduced MetO in oxidized protein was experimentally demonstrated [9,10,19,20]. On the contrary, biochemical assays demonstrated that neither the fRMsr, BisC, nor TorZ/MtsZ can catalyze the reduction of MetO in proteins [15,16,32,33].…”

“…In this work, we evaluated the capacity of the R. sphaeroides DorA DMSO reductase to reduce model oxidized proteins and MetO-containing peptides, and compared it to its ability to reduce DMSO and the free L-Met-R,S-O. The first oxidized protein tested was the bovine β-casein, an intrinsically disordered protein possessing 6 Met oxidizable as either Met-R-O or Met-S-O upon reaction with hydrogen peroxide and previously used for the characterization of several Msr enzymes [14,20]. We also showed that DorA can reduce MetO in two R. sphaeroides periplasmic proteins, TakP and PCu A C, both potentially exposed to oxidative conditions in the bacteria.…”