Characterization of the Methionine Sulfoxide Reductase Activities of PILB, a Probable Virulence Factor from Neisseria meningitidis

Abstract

PILB has been described as being involved in the virulence of bacteria of Neisseria genus. The PILB protein is composed of three subdomains. In the present study, the central subdomain (PILB-MsrA), the C terminus subdomain (PILB-MsrB), and the fused subdomain (PILB-MsrA/ MsrB) of N. meningitidis were produced as folded entities. The central subdomain shows a methionine sulfoxide reductase A (MsrA) activity, whereas PILB-MsrB displays a methionine sulfoxide reductase B (MsrB) activity. The catalytic mechanism of PILB-MsrB can be divided into two steps: 1) an attack of the Cys-494 on the sulfur atom of the sulfoxide substrate, leading to formation of a sulfenic acid intermediate and release of 1 mol of methionine/mol of enzyme and 2) a regeneration of Cys-494 via formation of an intradisulfide bond with Cys-439 followed by reduction with thioredoxin. The study also shows that 1) MsrA and MsrB display opposite stereoselectivities toward the sulfoxide function; 2) the active sites of both Msrs, particularly MsrB, are rather adapted for binding proteinbound MetSO more efficiently than free MetSO; 3) the carbon C␣ is not a determining factor for efficient binding to both Msrs; and 4) the presence of the sulfoxide function is a prerequisite for binding to Msrs. The fact that the two Msrs exhibit opposite stereoselectivities argues for a structure of the active site of MsrBs different from that of MsrAs. This is further supported by the absence of sequence homology between the two Msrs in particular around the cysteine that is involved in formation of the sulfenic acid derivative. The fact that the catalytic mechanism takes place through formation of a sulfenic acid intermediate for both Msrs supports the idea that sulfenic acid chemistry is a general feature in the reduction of sulfoxides by thiols.Peptide methionine sulfoxide reductase (MsrA) 1 activity is described as being involved in the virulence of the pathogens Escherichia coli, Streptococcus pneumoniae, Erwinia chrysanthemi, Mycoplasma genitalium, and Neisseria gonorrhoeae (1-4). Inspection of the alignment of the corresponding protein sequences shows that all possess in common a sequence that displays an MsrA activity. This MsrA activity has now been well characterized at the structural level (5, 6) and the enzymatic level (7). In particular, a sulfenic acid intermediate has been shown to be formed on Cys-51 of E. coli MsrA during the reduction of the sulfoxide function of methionine sulfoxide (MetSO). The active site can be represented as an open basin in which Cys-51, located at the N terminus of an ␣-helix, is accessible. Compared with the E. coli MsrA, the MsrAs from S. pneumoniae and from N. meningitidis or N. gonorrhoeae (called PILB) contain, in addition, an extension at the C terminus and at the C and N termini, respectively. This raised the question of the role of these extensions, in particular of the C-terminal extension. Sequence comparisons of the C-extension of PILB show amino acid identities with open reading frames of which no function has been...

Kinetic Characterization of the Chemical Steps Involved in the Catalytic Mechanism of Methionine Sulfoxide Reductase A from Neisseria meningitidis

“…MsrA from N. meningitidis possesses two cysteines at position 51 and 198 (13). As mentioned in the Introduction, the catalytic mechanism of the MsrA from N. meningitidis involves three chemical steps: a) the reduction of MetSO, which leads to the formation of a sulfenic acid intermediate on Cys-51 and a concomitant release of 1 mol of Met/mol of enzyme; b) the formation of an intradisulfide bond between Cys-51 and Cys-198; and c) the reduction of the disulfide bond by Trx, which finally liberates MsrA and Trx under reduced and oxidized forms, respectively.…”

“…The molecular concentration was determined spectrophotometrically, using extinction coefficient at 280 nm of 26,200 M Ϫ1 cm Ϫ1 for wild-type and C198S MsrA (13) and 20,480 M Ϫ1 cm Ϫ1 for W53F MsrA. In this report, N. meningitidis MsrA amino acid numbering is based on E. coli MsrA.…”

“…Site-directed Mutagenesis, Production, and Purification of Wild-type and Mutant N. meningitidis MsrAs-The E. coli strain used for all N. meningitidis MsrA productions was BE002 (MG1655 msrA::spec⍀, msrB::␣ 3kana), transformed with the plasmidic construction pSKPILBMsrA containing the coding sequence of msrA under the lac promoter (13). The BE002 strain was kindly provided by Dr F. Barras.…”

“…Storage of the enzymes was done as described previously (13). The molecular concentration was determined spectrophotometrically, using extinction coefficient at 280 nm of 26,200 M Ϫ1 cm Ϫ1 for wild-type and C198S MsrA (13) and 20,480 M Ϫ1 cm Ϫ1 for W53F MsrA.…”

“…Although MsrAs from Escherichia coli, Bos taurus, and Mycobacterium tuberculosis and MsrB from Neisseria gonohorreae show quite different three-dimensional x-ray structures and are specific for the S and the R isomers of the sulfoxide of MetSO, respectively, both structural classes of Msrs present a similar, new catalytic mechanism that includes a minimum of three chemical steps (Scheme 1). This is the case for MsrA and MsrB from Neisseria meningitidis (13). In the first step, a Michaelis complex is formed between Msr and MetSO that precedes the nucleophilic attack of the catalytic cysteine on the sulfoxide of MetSO.…”