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M Rangarajan

Publications and source records attributed to M Rangarajan.

At least 37 records · Page 2Linked to original sources
PubMed1997

The Tla protein of Porphyromonas gingivalis W50: a homolog of the RI protease precursor (PrpRI) is an outer membrane receptor required for growth on low levels of hemin.

The prpR1 gene of Porphyromonas gingivalis W50 encodes the polyprotein precursor (PrpRI) of an extracellular arginine-specific protease. PrpRI is organized into four distinct domains (pro, alpha, beta, and gamma) and is processed to a heterodimeric protease (RI) which comprises the alpha and beta components in a noncovalent association. The alpha component contains the protease active site, whereas the beta component appears to have a role in adherence and hemagglutination processes. DNA sequences homologous to the coding region for the RI beta component are present at multiple loci on the P. gingivalis chromosome and may represent a family of related genes. In this report, we describe the cloning, sequence analysis, and characterization of one of these homologous loci isolated in plasmid pJM7. The 6,041-bp P. gingivalis DNA fragment in pJM7 contains a major open reading frame of 3,291 bp with coding potential for a protein with an Mr 118,700. An internal region of the deduced sequence (V304 to N768) shows 98% identity to the beta domain of PrpRI, and the recombinant product of pJM7 is immunoreactive with an antibody specific to the RI beta component. The N terminus of the deduced sequence has regional similarity to TonB-linked receptors which are frequently involved in periplasmic translocation of hemin, iron, colicins, or vitamin B12 in other bacteria. We have therefore designated this gene tla (TonB-linked adhesin). In contrast to the parent strain, an isogenic mutant of P. gingivalis W50 in which the tla was insertionally inactivated was unable to grow in medium containing low concentrations of hemin (<2.5 mg liter(-1)), and hemin-depleted cells of this mutant failed to respond to hemin in an agar diffusion plate assay. These data suggest a role for this gene product in hemin acquisition and utilization. Furthermore, the mutant produced significantly less arginine- and lysine-specific protease activities than the parent strain, indicating that there may be a regulatory relationship between tla and other members of this gene family.

Adhesins, Bacterial↗
PubMed1996

Characterization of an adherence and antigenic determinant of the ArgI protease of Porphyromonas gingivalis which is present on multiple gene products.

This study was performed to characterize the antigen(s) recognized by a panel of monoclonal antibodies (MAbs) produced to be specific for Porphyromonas gingivalis whole cells which we had previously shown to bind to epitopes recognized by sera from periodontitis patients. Preliminary data had suggested that the arginine-specific proteases of P. gingivalis (ArgI, ArgIA, and ArgIB) contained the antigenic determinants of four of these antibodies (MAbs 1A1, 2B/H9, 7D5, and 3B1). The location of the binding sites was examined with purified P. gingivalis enzymes and recombinant regions of the ArgI polyprotein expressed by subclones of the prpR1 gene in Escherichia coli XL-1 Blue cells. All four antibodies were reactive with protein determinants within the beta subunit, a hemagglutinin and/or adhesin component, of the ArgI dimer. MAb 1A1 strongly inhibited the agglutination of human erythrocytes by P. gingivalis W50 culture supernatant, suggesting that the binding site for this antibody contains residues which are critical for the interaction with the erythrocyte surface. The determinant for MAb 1A1 was examined further by construction of a set of truncated forms of the beta component expressed as fusion proteins with glutathione S-transferase at the N terminus. Analysis of these constructs mapped the binding site for MAb 1A1 to PrpRI residues G-907 to T-931, GVSPKVCKDV TVEGSNEFAP VQNLT. Western blot (immunoblot) analysis of P. gingivalis whole-cell proteins demonstrated that MAb 1A1 reacts with several proteins in the Mr range of 20,000 to 120,000. Furthermore, an oligonucleotide probe corresponding to the coding sequence for the region of the ArgI beta component containing the MAb 1A1 binding site hybridized to multiple bands on genomic digests of P. gingivalis DNA. These data indicate that the MAb 1A1 epitope may be a component of a binding domain common to multiple gene products of this organism and may thus represent a functionally important target of the host's specific immune response to P. gingivalis in periodontal disease.

Adhesins, Bacterial↗
PubMed1995

Characterization, genetic analysis, and expression of a protease antigen (PrpRI) of Porphyromonas gingivalis W50.

Previous studies of the serum immunoglobulin G antibody response of periodontal patients have demonstrated significant reactivity to a cell surface or extracellular arginine-specific protease of Porphyromonas gingivalis which migrates as an approximately 50-kDa band on sodium dodecyl sulfate-polyacrylamide gels. In the present report, two forms of the enzyme (ArgI and ArgIA) with this electrophoretic behavior were isolated. ArgI is a heterodimer of alpha and beta subunits, and ArgIA is a monomer composed of the catalytically active alpha component alone. The gene encoding ArgI (prpR1 encoding protease polyprotein ArgI) was cloned from Sau3AI digests of P. gingivalis W50 DNA into pUC18. Sequence analysis demonstrated that the alpha and beta components are contiguous on the initial translation product and are flanked by large N- and C-terminal extensions. prpR1 is 97.5% identical to the rgp-1 gene from P. gingivalis H66. prpR1 expression in Escherichia coli demonstrated the presence of an internal transcription-translation initiation site which could permit independent expression of different regions of the polyprotein. Immunochemical analysis of P. gingivalis mid-logarithmic-phase cultures suggested that the processing of PrpRI may be closely coupled to its synthesis, with only the final stages taking place at the cell surface. Southern hybridization studies demonstrated that the prpR1 gene is widely distributed in other P. gingivalis strains and that a second homologous locus to the alpha component and at least two other homologous loci to the beta component are present on the P. gingivalis chromosome. These data indicate that the ArgI protease of P. gingivalis is a member of a family of sequence-related gene products which may share both functional and antigenic properties.

Adhesins, Bacterial↗
PubMed1993

Arthrobacter D-xylose isomerase: chemical modification of carboxy groups and protein engineering of pH optimum.

To try to lower the pH optimum, the carboxy groups of Arthrobacter D-xylose isomerase were coupled to glycinamide using a water-soluble carbodi-imide. In conditions that substituted all of the 59 carboxy groups in the denatured monomer, a maximum of 30 groups/monomer reacted in the native enzyme, whether in presence or absence of ligands, and the enzyme remained fully active and tetrameric throughout the coupling reaction. Purification by f.p.l.c. ion-exchange chromatography gave broad symmetrical peaks with increased pI, suggesting that the modified enzymes are essentially homogeneous. However, they are less stable than native enzyme in 8 M urea or on heating ('melting points' of 59 degrees versus 73 degrees C for the apoenzymes and 67 degrees versus 81.5 degrees C for the Mg(2+)-enzymes). Kinetic studies of the D-fructose isomerase activity at 30 degrees C showed that the glycinamidylated enzyme had unaltered activation constant for Mg2+, and Km was also similar to that of the native enzyme at pH 7.3, but increased rapidly at higher pH rather than remaining constant. Vmax. was constant from pH 6.2 to 8.0, suggesting a reduced pKa for His-219, which controls Vmax. in the native enzyme (normally 6.0). Three mutants were constructed by protein engineering with a view to reducing the pH optimum of enzyme activity. Two of these, Glu140-->Lys and Asp189-->Lys, could be detected in crude extracts of Escherichia coli by SDS/PAGE, but could not be purified, whereas mutant Trp136-->Glu was produced as a tetramer in amounts similar to the wild-type enzyme. However, it did not show any enzyme activity and was less stable in 0-9 M urea gradient PAGE.

Aldose-Ketose Isomerases↗
PubMed1993

Arthrobacter D-xylose isomerase: protein-engineered subunit interfaces.

Mutants of Arthrobacter D-xylose isomerase were constructed in which one or two disulphide bridges or additional salt bridges were introduced at the A-A* subunit interfaces. These showed no change in enzyme activity or stability compared with the wild-type enzyme. However, a Tyr253 mutant in which a disulphide bridge was introduced at the A-B* subunit interface showed reduced thermostability that was identical in both oxidized and reduced forms, and also reduced stability in urea. X-ray-crystallographic analysis of the Mn(2+)-xylitol form of oxidized Y253C (the Tyr253-->Cys mutant) showed a changed conformation of Glu185 and also alternative conformations for Asp254, which is a ligand to the Site-[2] metal ion. With fructose, Mg(2+)-Y253C has a similar Km to that of the wild-type, and its Vmax. is also similar below pH 6.4, but declined thereafter. In the presence of Co2+, Y253C has lower activity than wild-type at all pH values, but its activity also declines at alkaline pH. These results suggest that electrostatic repulsion from the new position of Glu185 causes Asp254 to move when His219 is unprotonated, thereby preventing M2+ binding at Site [2]. These results also suggest that subunit dissociation does not lie on the pathway of thermal inactivation of D-xylose isomerase, but that movements of active-site groups are a trigger for conformational changes that initiate the unfolding process.

Aldose-Ketose Isomerases↗
PubMed1993

Arthrobacter D-xylose isomerase: partial proteolysis with thermolysin.

The pattern and kinetics of partial proteolysis of Arthrobacter D-xylose isomerase tetramer was studied in order to determine the flexibility of surface loops that may control its stability. It was completely resistant to trypsin, chymotrypsin and elastase at 37 degrees C, but thermolysin cleaved specifically and quantitatively at Thr-347-Leu-348 between helices 10 and 11 to remove 47 residues from the C-terminus of each 43.3 kDa subunit. At high temperatures, helices 9 and 10 were removed from each 38 kDa subunit to give a 36 kDa tetramer. The kinetics of nicking by thermolysin indicated that the Thr-347-Leu-348 loop is locked at low temperatures, but 'melts' at 25 degrees C and is fully flexible above 34 degrees C. The flexibility appears to be associated with binding of Ca2+ ions at the active site, since Co2+, Mg2+ and xylitol protect in proportion to their ability to displace Ca2+. The missing C-terminal helices make many intersubunit contacts that appear in the structure to stabilize the tetramer, but the properties of the purified nicked proteins are almost indistinguishable from the native enzyme. Both the 38 kDa tetramer and the 36 kDa tetramer are identically active and dissociate similarly in urea or SDS to fully active dimers, but the nicked dimers are slightly less stable to urea at 62 degrees C. In the Mg2+ form the thermostability of the 38 kDa tetramer is identical with that of the native enzyme, but the 36 kDa tetramer has a slightly lower 'melting point' (70 degrees C versus 80 degrees C), which may be due to unravelling from the end of helix 8. Since elimination of all the C-terminal helices and many intersubunit contacts has so little effect, one can conclude that the 'weak point' that controls the protein's thermostability lies within the N-terminal beta-barrel domain.

Aldose-Ketose Isomerases↗
PubMed1992

Stability of Arthrobacter D-xylose isomerase to denaturants and heat.

There was no inactivation of Mg(2+)-containing Arthrobacter D-xylose isomerase up to 1 h in 0-8 M-urea at 22 degrees C, but over this range there was rapid reversible dissociation into fully active dimers with a midpoint around 4 M-urea, as shown by gradient urea gels with an activity stain, and by ion-exchange chromatography and gel filtration in urea buffers. These dimers must have the A-B* conformation, since the tetramer could dissociate into A-A*, A-B or A-B* dimer conformations, but only residues across the A-B* interface contribute to the active site. The kinetics of inactivation of the Mg(2+)-containing enzyme in 8 M-urea at higher temperatures suggest a partially unfolded Mg-A-B* dimer intermediate with 50% activity, followed by irreversible inactivation coincident with the appearance of unfolded monomer. In 0-4 M guanidinium chloride, a similar reversible dissociation into active dimers occurs, but activity falls, suggesting that A-A* and/or A-B dimers might be part of the mixture. Low concentrations of SDS also give active dimers leading to unfolded monomers, but SDS above 1% (w/v) provides relative stabilization. The apoenzyme is least thermostable (t 1/2 at 80 degrees C, pH 7, = 0.06 h) but Mg2+ stabilizes strongly (t 1/2 = 5.5 h) and Co2+ even more so. Competitive inhibitors or substrates provide a small further stabilization, but this effect is more marked at 80 degrees C, pH 5.5. Together with a marked decrease in optimum pH with temperature, this allows batch isomerizations of glucose under these conditions that produce clean but sweeter syrups.

Aldose-Ketose Isomerases↗
PubMed1992

Mechanism of D-fructose isomerization by Arthrobacter D-xylose isomerase.

The mechanism of D-fructose isomerization by Arthrobacter D-xylose isomerase suggested from X-ray-crystallographic studies was tested by detailed kinetic analysis of the enzyme with various metal ions at different pH values and temperatures. At D-fructose concentrations used in commercial processes Mg2+ is the best activator with an apparent dissociation constant of 63 microM; Co2+ and Mn2+ bind more strongly (apparent Kd 20 microM and 10 microM respectively) but give less activity (45% and 8% respectively). Ca2+ is a strict competitive inhibitor versus Mg2+ (Ki 3 microM) or Co2+ (Ki 105 microM). The kinetics show a compulsory order of binding; Co2+ binds first to Site 2 and then to Site 1; then D-fructose binds at Site 1. At normal concentrations Mg2+ binds at Site 1, then D-fructose and then Mg2+ at Site 2. At very high Mg2+ concentrations (greater than 10 mM) the order is Mg2+ at Site 1, Mg2+ at Site 2, then D-fructose. The turnover rate (kcat.) is controlled by ionization of a residue with apparent pKa at 30 degrees C of 6.0 +/- 0.07 (Mg2+) or 5.3 +/- 0.08 (Co2+) and delta H = 23.5 kJ/mol. This appears to be His-219, which is co-ordinated to M[2]; protonation destroys isomerization by displacing M[2]; Co2+ binds more strongly at Site 2 than Mg2+, so competes more strongly against H+. The inhibition constant (Ki) for the two competitive inhibitors 5-thio-alpha-D-glucopyranose and D-sorbitol is invariant with pH, but Km(app.) in the Mg[1]-enzyme is controlled by ionization of a group with pKa 6.8 +/- 0.07 and delta H = 27 kJ/mol, which appears to be His-53. This shows that Km(app.) is a complex constant that includes the rate of the ring-opening step catalysed by His-53, which explains the pH-dependence. In the Mg[1]Mg[2]-enzyme or Co[1]Co[2]-enzyme, the pKa is lower (6.2 +/- 0.1 or 5.6 +/- 0.08) because of the extra adjacent cation. Hence the results fit the previously proposed pathway, but show that the mechanisms differ for Mg2+ and Co2+ and that the rate-limiting step is isomerization and not ring-opening as previously postulated.

Aldose-Ketose Isomerases↗
PubMed1991

D-Xylose (D-glucose) isomerase from Arthrobacter strain N.R.R.L. B3728. Purification and properties.

D-Xylose (D-glucose) isomerase was purified to homogeneity in yields of approx. 1 g/kg of wet cells from a strain of Arthrobacter that produces it as about 10% of total soluble protein. It is a tetramer of identical 43,114 Da subunits containing a preponderance of acidic residues and no cysteine. Partial protein sequences were determined as a step to gene cloning. It requires Mg2+, Co2+ or Mn2+ for activity, Mg2+ being best; Ca2+ is an inhibitor, competitive with Mg2+. It is a good D-glucose isomerase with kcat. 1200 min-1 at pH 8 at 60 degrees C, which is higher than that of any other enzyme of this class. L-Arabinose, D-ribose and D-lyxose are poor substrates, with kcat. 78, 31 and 3.7 min-1 respectively at pH 8 at 30 degrees C, compared with 533 min-1 for D-xylose. Xylitol is a true competitive inhibitor for D-xylose (Ki 0.3 mM), but D-sorbitol shows mixed inhibition (Ki 6.5 mM). For D-fructose the pH optimum at 60 degrees C is 8, and at pH 7 the Arrhenius activation energy is 75 kJ/mol over the range 30-70 degrees C.

Aldose-Ketose Isomerases↗
PubMed1984

Occurrence of a Lysogenic Streptomyces sp. on the Nodule Surface of Black Gram (Vigna mungo (L.) Hepper).

A lysogenic Streptomyces sp., strain NS.A4, which was isolated from the nodule surface of black gram (Vigna mungo (L.) Hepper), was found to inhibit rhizobia of fast-and slow-growing strains of cowpeas and soybeans. It exhibited plaques when there was a change in cultural conditions. Repeated culturing of the organism in nutrient agar and broth confirmed the infection of Streptomyces sp. strain NS.A4 by an actinophage. Addition of the culture filtrate of Streptomyces sp. strain NS.A4 to shaken broth cultures of three other Streptomyces spp. resulted in phage infection.

Journal Article↗
PubMed1976

Studies on sequencing of peptides from the carboxyl terminus by using the thiocyanate method.

We report on our experience in applying the thiocyanate method developed by Stark (1968) (Biochemistry 7, 1796-1807) to the sequencing of short peptides from the carboxyl end in free solution. Yields fell to very low levels after three cycles of degradation. The method was time-consuming because of the filtration and freeze-drying stages involved. To overcome these problems, peptides were attached to modified polystyrene polymers for sequential degradation in the solid phase, and a maximum of six amino acids was determined. Also, ribonuclease was attached to active-ester glass beads and sequential degradation was carried out to determine six amino acids at the C-terminal end of this protein.

Amino Acid Sequence↗