Surface components of Porphyromonas gingivalis.

BACKGROUND AND OBJECTIVE: Research on Porphyromonas gingivalis, a periodontopathogen, has provided a tremendous amount of information over the last 20 years, which may exceed in part than that on other closely related members in terms of phylogenetic as well as proteomic criteria, including Bacteroides fragilis and B. thetaiotaomicron as major anaerobic, opportunistic pathogens in the medical field. In this minireview, we focused on recent research findings concerning surface components such as outer membrane proteins and fimbriae, of P. gingivalis. MATERIAL AND METHODS: Elucidation of the surface components in P. gingivalis was especially difficult because outer membrane proteins are tightly bound to lipopolysaccharide and they are resistant to dissociation and separation from each other, even during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, unless samples are appropriately heated. In addition, P. gingivalis is asaccharolytic and therefore a potent proteolytic bacterium, another factor causing difficulty in research. The study of the surface components was carefully carried out considering these unique features in P. gingivalis when compared with other gram-negative bacteria, including Escherichia coli and Pseudomonas aeruginosa. RESULTS: Separation of outer membrane proteins, and characterization of OmpA-like proteins and RagAB as major proteins, is described herein. Our recent findings on FimA and Mfa1 fimbriae, two unique appendages in this organism, and on their regulation of expression are also described briefly. CONCLUSION: Surface components of P. gingivalis somehow have contact with host tissues and cells because of the outermost cell elements. Therefore, such bacterial components are potentially important in the occurrence of periodontal diseases.

A multinuclear NMR study of the active site of an endoglucanase from a strain of Bacillus. Use of Trp residues as structural probes.

In the hydrolytic reaction catalyzed by an endoglucanase from a Bacillus strain (endoglucanase K), 2 of 12 Trp residues, Trp174 and Trp243, are responsible for binding of the substrate and/or for the catalysis (Kawaminami, S., Ozaki, K., Sumitomo, N., Hayashi, Y., Ito, S., Shimada, I., and Arata, Y. (1994) J. Biol. Chem. 269, 28752-28756). Here we report results of a stable isotope-aided NMR analysis of the active site of endoglucanase K, using Trp174 and Trp243 as structural probes. Hydrogen-deuterium exchange experiments performed for the NH protons of main and side chains of Trp residues revealed that Trp174 and Trp243 are located in the hydrophilic and hydrophobic microenvironments in the active site, respectively. We also carried out pH titration experiments for indole C2 proton resonances of Trp residues and measured the pH dependence of specific activities for wild-type endoglucanase K and its mutants in which Glu or Asp residues are replaced with their respective amide forms. On the basis of the results obtained from the present study, we conclude that (a) Glu130 and Asp191, which are in spatial proximity to Trp174 and Trp243 in the active site, play a crucial role in the enzymatic activity; (b) Glu130 and Asp191 interact with each other in the active site, leading to an increase in the pKa values to 5.5 for both amino acid residues; and (c) the pKa values of Glu130 and Asp191 would lead to an unusually narrow pH-activity profile of the endoglucanase K.

Amino acid sequence and stereoselective hydrolytic reaction of an endo-1,4-beta-glucanase from a Bacillus strain.

Bacillus sp. KSM-522 produces three different extracellular endo-1,4-beta-glucanases [EGs; Okoshi et al., Agric. Biol. Chem., 54, 83-89 (1990)]. Here, we report the molecular cloning and sequencing of the gene for the fourth EG (EG-IV) of the organism and the mechanism of its hydrolytic reaction. The structural gene contained an open reading frame of 1911 bp, corresponding to 636 amino acids, the amino acid sequence of which was very close to that of an EG of Clostridium cellulovorans, belonging to the cellulase family E2. The molecular mass of the extracellular mature enzyme (Ser26 through Lys636) was calculated to be 69,076 Da, a value close to the 69.2 kDa measured for the recombinant EG-IV expressed in Bacillus subtilis. The optimum pH and temperature for activity of the recombinant enzyme were pH 8.0 and 50 degrees C, respectively. By 1H-NMR spectroscopy, we demonstrated that the hydrolysis of p-nitrophenyl beta-D-cellotrioside by EG-IV proceeded with inversion of the anomeric configuration.

Application of the upstream region of a Bacillus endoglucanase gene to high-level expression of foreign genes in Bacillus subtilis.

A 0.4-kb ScaI-HpaI fragment, 199 bp upstream of the structural gene for alkaline endoglucanase, from the alkalophilic Bacillus sp. KSM-64, was found to be essential for the extracellular production of the enzyme by recombinant Bacillus subtilis cells. We constructed a new vector, pHSP64 (5.5 kb), using pHY300PLK and part of the 5' region of the endoglucanase that contained a possible promoter region. Using recombinant B. subtilis cells that carried this vector, very high production of two endoglucanases and of chloramphenicol acetyltransferase was done.

Stereoselective hydrolysis catalyzed by a Bacillus endoglucanase in family D.

An endoglucanase in family D, purified from a strain of Bacillus, was found to catalyze the hydrolysis of p-nitrophenyl beta-D-cellotrioside to generate alpha-cellobiose, as determined by 1H-NMR spectroscopy. The hydrolysis of the beta-1,4 glucosidic bond by the enzyme proceeds, therefore, by an inversion mechanism. Furthermore, the interconversion of the alpha- and beta-anomeric protons in the products of hydrolysis, after equilibrium had been reached by mutarotation, was directly characterized by magnetization transfer NMR experiment that exploited the truncated driven nuclear Overhauser effect.

A stable isotope-aided NMR study of the active site of an endoglucanase from a strain of Bacillus.

Heteronuclear single-quantum coherence two-dimensional NMR spectroscopy has been used to investigate the active site of endoglucanase K (46 kDa) from Bacillus sp. KSM-330, in which Trp are important for expression of the activity. Endoglucanase K, which was specifically labeled with [indole-2-13C]Trp, was prepared from recombinant Bacillus subtilis that carried the gene for this enzyme on an expression vector, pHSP-KC331. Twelve cross-peaks originating from the C-2 position of Trp residues of endoglucanase K were separately observed in 1H-13C heteronuclear single-quantum coherence spectrum, and six of the cross-peaks have been assigned site-specifically by using site-directed mutagenesis. The chemical shifts of the cross-peaks originating from Trp-174 and Trp-243 were affected by the addition of cellotriose that was used as a competitive inhibitor of the enzyme. On the basis of the NMR data obtained after chemical modification of the enzyme by N-bromosuccinimide, it appears that Trp-174 was oxidized first with retention of 56% of the original activity and Trp-243 was then oxidized with complete loss of activity. Substitution of Trp-174 or Trp-243 by Tyr residue caused a decrease in the specific activity of the enzyme to 49 or 8% of that of the wild-type enzyme, respectively. Km values of these mutant enzymes for p-nitrophenyl beta-D-cellotrioside increased to 5 and 8 times those of the wild-type enzyme, respectively, while kcat values of both of the mutant enzymes decreased to one-fifth of those of the wild-type enzymes. These results suggest that Trp-174 and Trp-243 play an important role in binding of the substrate and/or in the catalytic activity.

Multinuclear NMR study of the structure of the Fv fragment of anti-dansyl mouse IgG2a antibody.

A multinuclear NMR study is reported of Fv, which is a minimum antigen-binding unit of immunoglobulin. Fv has been prepared by clostripain digestion of a mouse anti-dansyl IgG2a monoclonal antibody that lacks the entire CH1 domain [Takahashi, H., Igarashi, T., Shimada, I., & Arata, Y. (1991) Biochemistry 30, 2840-2847]. A variety of Fv analogues labeled with 2H in the aromatic rings and with 13C and/or 15N in the peptide bonds have been prepared and used for multinuclear NMR analyses of Fv in the absence and presence of epsilon-dansyl-L-lysine (DNS-Lys). It has been shown that 1H-15N shift correlation spectra of Fv sensitively reflect the antigen binding and can be used along with 1H and 13C spectral data for the structural analyses of antigen-antibody interactions. Hydrogen-deuterium exchange of the amide protons has been followed in the absence and presence of DNS-Lys by using the 1H-15N shift correlation spectra. Use of the beta-shift observed for the carbonyl carbon resonances has also been helpful in following the hydrogen-deuterium exchange. On the basis of the NMR data obtained, the static and dynamic structure of the Fv fragment in the absence and presence of DNS-Lys has been discussed.