Voltammetry of cytochrome P450 (cyt P450) enzymes in ultrathin films with polyions was related for the first time to electronic and secondary structure. Heterogeneous electron transfer (hET) rate constants for reduction of the cyt P450s depended on heme iron spin state, with low spin cyt P450cam giving a value 40-fold larger than high spin human cyt P450 1A2, with mixed spin human P450 cyt 2E1 at an intermediate value. Asymmetric reduction–oxidation peak separations with increasing scan rates were explained by simulations featuring faster oxidation than reduction. Results are consistent with a square scheme in which oxidized and reduced forms of cyt P450s each participate in rapid conformational equilibria. Rate constants for oxidation of ferric cyt P450s in films by t-butyl hydroperoxide to active ferryloxy cyt P450s from rotating disk voltammetry suggested a weaker dependence on spin state, but in the reverse order of the observed hET reduction rates. Oxidation and reduction rates of cyt P450s in the films are also likely to depend on protein secondary structure around the heme iron.

c Rickettsia prowazekii, the etiologic agent of epidemic typhus, is a potential biological threat agent. Its outer membrane protein B (OmpB) is an immunodominant antigen and plays roles as protective envelope and as adhesins. The observation of the correlation between methylation of lysine residues in rickettsial OmpB and bacterial virulence has suggested the importance of an enzymatic system for the methylation of OmpB. However, no rickettsial lysine methyltransferase has been characterized. Bioinformatic analysis of genomic DNA sequences of Rickettsia identified putative lysine methyltransferases. The genes of the potential methyltransferases were synthesized, cloned, and expressed in Escherichia coli, and expressed proteins were purified by nickelnitrilotriacetic acid (Ni-NTA) affinity chromatography. The methyltransferase activities of the purified proteins were analyzed by methyl incorporation of radioactively labeled S-adenosylmethionine into recombinant fragments of OmpB. Two putative recombinant methyltransferases (rRP789 and rRP027-028) methylated recombinant OmpB fragments. The specific activity of rRP789 is 10-to 30-fold higher than that of rRP027-028. Western blot analysis using specific antibodies against trimethyl lysine showed that both rRP789 and rRP027-028 catalyzed trimethylation of recombinant OmpB fragments. Liquid chromatographytandem mass spectrometry (LC/MS-MS) analysis showed that rRP789 catalyzed mono-, di-, and trimethylation of lysine, while rRP027-028 catalyzed exclusively trimethylation. To our knowledge, rRP789 and rRP027-028 are the first biochemically characterized lysine methyltransferases of outer membrane proteins from Gram-negative bacteria. The production and characterization of rickettsial lysine methyltransferases provide new tools to investigate the mechanism of methylation of OmpB, effects of methylation on the structure and function of OmpB, and development of methylated OmpB-based diagnostic assays and vaccine candidates. Rickettsial OmpBs belong to the outer membrane protein autotransporter family and contain large passenger domains. Rickettsial OmpBs have been shown to participate in adhesion to mammalian cells in vitro (8,36). Ectopically expressed rickettsial OmpB in Escherichia coli has been demonstrated to be sufficient in mediating bacterial attachment and invasion of cultured mammalian cells (8,24). Unlike the ␣-helical transmembrane proteins in plasma membranes, the autotransporter domains of these outer membrane proteins have structural characteristics of ␤-barrel integral membrane proteins (3, 33). As the immunodominant antigenic surface protein, native OmpB induces strong humoral and cellular immune responses in animal models and in patients (6,12,15,16,23). Methylation of OmpA of Rickettsia canadensis and ompB of Rickettsia typhi is known, but whether this is important for any other pathogenic rickettsia is unknown.One important but not well understood structural feature of rickettsial OmpB is the methylation at ε-amino groups of lysine residues (28). Postt...

Background: Methylation of OmpB has been implicated in rickettsial virulence. Results: Native OmpBs purified from Rickettsia contain mono-and trimethyllysine at specific locations that coincide with those catalyzed by methyltransferases in vitro. Conclusion: The number of trimethyllysine clusters in OmpBs correlates with degree of virulence. Significance: This study provides new insight into methylation of OmpB and its correlation with virulence.

Rickettsia belong to a family of Gram-negative obligate intracellular infectious bacteria that are the causative agents of typhus and spotted fever. Outer membrane protein B (OmpB) occurs in all rickettsial species, serves as a protective envelope, mediates host cell adhesion and invasion, and is a major immunodominant antigen. OmpBs from virulent strains contain multiple trimethylated lysine residues, whereas the avirulent strain contains mainly monomethyllysine. Two protein-lysine methyltransferases (PKMTs) that catalyze methylation of recombinant OmpB at multiple sites with varying sequences have been identified and overexpressed. PKMT1 catalyzes predominantly monomethylation, whereas PKMT2 catalyzes mainly trimethylation. Rickettsial PKMT1 and PKMT2 are unusual in that their primary substrate appears to be limited to OmpB, and both are capable of methylating multiple lysyl residues with broad sequence specificity. Here we report the crystal structures of PKMT1 from Rickettsia prowazekii and PKMT2 from Rickettsia typhi, both the apo form and in complex with its cofactor S-adenosylmethionine or S-adenosylhomocysteine. The structure of PKMT1 in complex with S-adenosylhomocysteine is solved to a resolution of 1.9 Å. Both enzymes are dimeric with each monomer containing an S-adenosylmethionine binding domain with a core Rossmann fold, a dimerization domain, a middle domain, a C-terminal domain, and a centrally located open cavity. Based on the crystal structures, residues involved in catalysis, cofactor binding, and substrate interactions were examined using sitedirected mutagenesis followed by steady state kinetic analysis to ascertain their catalytic functions in solution. Together, our data reveal new structural and mechanistic insights into how rickettsial methyltransferases catalyze OmpB methylation.Methylation of outer membrane proteins has been implicated in rickettsial virulence. Rickettsia belong to a family of obligatory intracellular infectious bacteria that are the causative agents of typhus and spotted fever (1). The bacterial outer membrane of Rickettsia contains a major surface protein called OmpB 5 that occurs in all rickettsial species and accounts for up to 15% of total cellular proteins (2-4). OmpB has been shown to mediate host cell adhesion, attachment, and invasion (5-7). OmpB belongs to the family of autotransporters (8) and is a major immunodominant antigen (3). The precursor of OmpB consists of a signal peptide, a passenger domain, and a C-terminal ␤-barrel domain (9). The passenger domain of OmpB has been shown to undergo methylation at its lysine residues, and this methylation appears to be associated with rickettsial pathogenicity and immunogenic response (10 -15). OmpB was first found to be methylated by amino acid composition analysis and later confirmed using mass spectrometric methods. The levels of methylation of OmpB from several virulent and avirulent strains appear to correlate well with the virulence level of the strains. In-depth analysis of methylation profiles, using semiqu...