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Comparative Study
. 2007 Jun 5;104(23):9597-602.
doi: 10.1073/pnas.0703774104. Epub 2007 May 29.

Free methionine-(R)-sulfoxide Reductase From Escherichia Coli Reveals a New GAF Domain Function

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Free PMC article
Comparative Study

Free methionine-(R)-sulfoxide Reductase From Escherichia Coli Reveals a New GAF Domain Function

Zhidong Lin et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

The reduction of methionine sulfoxide (MetO) is mediated by methionine sulfoxide reductases (Msr). The MsrA and MsrB families can reduce free MetO and MetO within a peptide or protein context. This process is stereospecific with the S- and R-forms of MetO repaired by MsrA and MsrB, respectively. Cell extracts from an MsrA(-)B(-) knockout of Escherichia coli have several remaining Msr activities. This study has identified an enzyme specific for the free form of Met-(R)-O, fRMsr, through proteomic analysis. The recombinant enzyme exhibits the same substrate specificity and is as active as MsrA family members. E. coli fRMsr is, however, 100- to 1,000-fold more active than non-selenocysteine-containing MsrB enzymes for free Met-(R)-O. The crystal structure of E. coli fRMsr was previously determined, but no known function was assigned. Thus, the function of this protein has now been determined. The structural similarity of the E. coli and yeast proteins suggests that most fRMsrs use three cysteine residues for catalysis and the formation of a disulfide bond to enclose a small active site cavity. This latter feature is most likely a key determinant of substrate specificity. Moreover, E. coli fRMsr is the first GAF domain family member to show enzymatic activity. Other GAF domain proteins substitute the Cys residues and others to specifically bind cyclic nucleotides, chromophores, and many other ligands for signal potentiation. Therefore, Met-(R)-O may represent a signaling molecule in response to oxidative stress and nutrients via the TOR pathway in some organisms.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Sequence of E. coli fRMsr. Six tryptic fragments were identified by mass spectrometry from E. coli fRMsr purified from MsrAB cells. The peptides are indicated in bold, underlined, and labeled 1–6. The identified sequence corresponds to the hypothetical E. coli protein NP_288269 and E. coli yebR of unknown function.
Fig. 2.
Fig. 2.
Expression construct and purification of recombinant E. coli fRMsr. (A) N-terminal sequence for the expression construct. Expression from this modified pET19 vector yields a 24-kDa species containing an N-terminal, His10-tag with intervening PreScission protease and enterokinase cleavage sites. The protein sequence from the second Met corresponds to the full-length E. coli fRMsr in Fig. 1. Initiation of protein synthesis at the third Met would result in a protein 18 kDa long. (B) SDS/PAGE and Western blot analysis of purified 18- and 24-kDa fRMsr species. Lanes 1 and 3, 18-kDa fRMsr; lane 2 and 4, 24-kDa fRMsr. An anti-His tag antibody (Qiagen, Valencia, CA) positively labels only the 24-kDa fRMsr.
Fig. 3.
Fig. 3.
Biochemical characterization of E. coli fRMsr. (A) Substrate specificity analysis of E. coli fRMsr. The rate observed for the control sample (no substrate) indicates the intrinsic NADPH oxidase rate of eTrxR. The reaction conditions were: fRMsr (0.102 μM), NADPH (250 μM), eTrxR (0.64 μM), eTrx (40 μM), bMsrA and ngMsrB (1.5 μM). The substrate concentration for the smaller substrates was 13 mM. For the peptide, a concentration of 26 mM was used, assuming that the proportion of Met-(R)-O and Met-(S)-O were equivalent. (B) Kinetic characterization of E. coli fRMsr. The reductase activity was assayed at various concentrations of purified Met-(R)-O. The reaction system included fRMsr (0.072 μM), NADPH (250 μM), and eTrxR (0.64 μM) and eTrx (2 μM, 7 μM, 30 μM; increasing in concentration from the bottom curve to the top).
Fig. 4.
Fig. 4.
E. coli fRMsr structure and comparisons with the GAF domains from yeast and Anabaena. (A) Overall fold of E. coli fRMsr. A Mes molecule (green carbon atoms; shown in stick rendering) is bound within an enclosed cavity (PDB code 1VHM) (17). (B) Close-up view of the Mes molecule and its proximity to three conserved Cys residues: Cys-84, Cys-94, and Cys-118. Surface loops 1 and 2 are linked together by a disulfide bond between Cys-84 and Cys-118. (C) Superposition of the Mes cavity residues for fRMsr (light blue carbon atoms) and the yeast YKG9 GAF domain (purple carbon atoms; PDB code 1F5M) (32). The first number in each label indicates the residue number for E. coli fRMsr. (D) Superposition of E. coli fRMsr and one of the GAF domains from Anabena adenylate cyclase (dark blue carbon atoms) complexed with cAMP (magenta carbon atoms; PDB code 1YKD) (41). Note the structural differences in loop 1 and in particular loop 2 of Anabaena which contains a α-helix. (E) Stereoview of the E. coli fRMsr and Anabaena cavities illustrating the lack of conservation of all residues, but the similar localization of the phosphate and sulfonate moieties.

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