Micrococcus Luteus Cells Research Articles

Proteins of the Rpf family: immune cell reactivity and vaccination efficacy against tuberculosis in mice.

It was shown recently that Mycobacterium tuberculosis expresses five proteins that are homologous to Rpf (resuscitation promoting factor), which is secreted by growing cells of Micrococcus luteus. Rpf is required to resuscitate the growth of dormant Micrococcus luteus organisms, and its homologues may be involved in mycobacterial reactivation. Mycobacterial Rpf-like products are secreted proteins, which makes them candidates for recognition by the host immune system and anti-Rpf immune responses potentially protective against reactivated tuberculosis. Here we report that the Rpf protein itself and four out of five of its mycobacterial homologues, which were administered as subunit vaccines to C57BL/6 mice, are highly immunogenic. Rpf-like proteins elicit immunoglobulin G1 (IgG1) and IgG2a responses and T-cell proliferation and stimulate production of gamma interferon, interleukin-10 (IL-10), and IL-12 but not IL-4 or IL-5. Both humoral and T-cell responses against these antigens show a high degree of cross-reactivity. Vaccination of mice with Rpf-like proteins results in a significant level of protection against a subsequent high-dose challenge with virulent M. tuberculosis H37Rv, both in terms of survival times and mycobacterial multiplication in lungs and spleens.

Fourier transform IR spectroscopic appraisal of radiation damage in Micrococcus luteus.

Fourier transform IR spectroscopy (FTIR) is used to analyze cells of Micrococcus luteus, the type species of the highly heterogeneous genus Micrococcus that belongs to the Micrococcaceae family. The cells of M. luteus, which is a Gram-positive and yellow-pigmented bacterium, are submitted to increasing doses of gamma radiation. Irradiation leads to the generation of reactive oxygen species that induce biochemical changes as shown in spectral profiles. Beyond a dose of 0.70 kGy, significant differences between samples are observed, particularly in the 1485-900 cm(-1) region, which contains information about membrane lipids, cell wall polysaccharides, and nucleic acids. After a dose of 16.50 kGy, M. luteus is reincubated for times ranging from 1 to 24 h. Postirradiation reincubated bacteria are found far from the control and irradiated cells (mainly in the 985-900 cm(-1) range), suggesting that a biomolecular rearrangement occurs as soon as reincubation begins in the growth medium. Thus, FTIR spectroscopy appears to be a very useful technique for the rapid visualization of the alterations induced by both the radiation and mutagenic response during reincubation. The use of mathematical methods gives good insight into the biomolecular compounds involved in these two mechanisms. In view of these preliminary results, we hypothesize that it can be successfully applied to any type of tissue and that it may be a future interesting tool for evaluating the effects of radiation in humans.

Formation of artificial, structured microbial consortia (ASMC) by dielectrophoresis

Artificial microbial consortia with defined internal microstructure (ASMC) were constructed by attracting cells of different microbial species to planar micro-electrodes by dielectrophoresis. Cells of Saccharomyces cerevisiae, Escherichia coli and Micrococcus luteus were deposited between the micro-electrodes sequentially to form layered cell aggregates with defined internal structures. The position of the different micro-organisms on the surface and within the cell layers was controlled by varying the electrode geometry, by introducing the different micro-organisms at different times, and by energising different parts of the electrode structure at different times. The cell arrays were stabilised using UV photopolymerised acrylamide-based hydrogels containing acrylamide and N, N′-methylene-bis acrylamide in the ratio of 15/2.2 (w/w), at a concentration of 5%. Although the viability of S. cerevisiae was only slightly affected after incubation in the monomer solution for 30 min, cells immobilised in the polymerised hydrogel exhibited reduced rates of glucose consumption and ethanol production compared with freely suspended cells, possibly due to diffusional limitation in the relatively thick hydrogels used. The gel-stabilised, structured consortia can either remain on the micro-electrode arrays as a structured biofilm, or they can be removed from the electrodes and used in a suspended form. The artificial, structured microbial consortia could be used to study microbial interactions and to create novel metabolic interactions between micro-organisms.

Enzymatic activity of PEOMA-modified lysozyme obtained from degradation of Micrococcus luteus cell and N-acetylated chitosan

A chicken egg white lysozyme was modified with a polyethyleneoxide-maleic anhydride (PEOMA) copolymer and two types of substrates, Micrococcus luteus (ML) cell walls and N-acetylated chitosan (NAC), which were used to determine the enzymatic activity of the PEOMA-modified lysozymes with a different degree of modification (DM). The ML cell walls and NAC were used as the substrates in their heterogeneous and homogeneous states, respectively. Modified lysozymes whose DM values ranged from 0 to 70% were used for both substrates. The enzymatic activity of the PEOMA-modified lysozyme drastically decreased at a DM from 40 to 50% in both cases, and an identical relationship between the enzymatic activity and the DM were observed. From these results, it is obvious that the enzymatic activity of PEOMA-modified lysozymes can be determined by using the two substrates, without considering the state of the substrate.

Induction of necrosis factor-alpha and interleukin-6 in mice in vivo and in murine peritoneal macrophages and human whole blood cells in vitro by Micrococcus luteus teichuronic acids.

Earlier studies showed that Micrococcus luteus cells and cell walls induced anaphylactoid reactions leading to death, in some instances within 1 h, in C3H/HeN mice primed with muramyl dipeptide (MDP). They also induced serum cytokines in the surviving mice. The present study investigated the structural components responsible for these activities. Teichuronic acids, a component of M. luteus cell walls, induced tumour necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) in MDP-primed C3H/HeN mice. Peptidoglycans had little effect on the cytokine-inducing activities. Reducing teichuronic acids, i.e., teichuronic acids whose carboxyl groups had been reduced, lost their cytokine-inducing activities. Neither peptidoglycans nor teichuronic acids induced anaphylactoid reactions in the MDP-primed mice. Purified teichuronic acids also induced TNF-alpha and IL-6 production in C3H/HeN murine peritoneal macrophages and human whole-blood cells in the culture, but reduced teichuronic acids did not. The purified teichuronic acids induced no TNF-alpha and only low levels of IL-6 in MDP-primed C3H/HeJ mice, and neither cytokine in peritoneal macrophage cultures from C3H/HeJ mice with a single point of mutation in Toll-like receptor 4 (TLR4) gene. These findings suggest that induction of cytokines by teichuronic acids is mainly TLR4-dependent.

Antibacterial activity of Bacillus amyloliquefaciens phage endolysin without holin conjugation

To characterize the enzymatic activity and antibacterial activity of endolysin encoded by a Bacillus amyloliquefaciens phage, the open reading frame encoding endolysin was amplified by PCR and cloned into the expression plasmid pET21d(+). The resultant plasmid was used to transform Escherichia coli JM109(DE3). Production of endolysin in the cytosol facilitated cell lysis without coproduction of holin, which is considered to degrade or alter the cytoplasmic membrane. The phage endolysin was overexpressed and purified. Although the specific activity of the purified phage endolysin towards lyophilized Micrococcus luteus cells was 1 11 of the activity of chicken egg white lysozymes, the endolysin showed stronger antibacterial activity towards E. coli W3110, E. coli JM109(DE3) and Pseudomonas aeruginosa PAO1 than chicken egg white lysozymes. The antibacterial activity of the endolysin towards these three bacterial strains was marked when EDTA was added to the endolysin solution.

Copper biosorption by chemically treated Micrococcus luteus cells

In order to clarify the binding states of copper in microbial cells, copper biosorption from aqueous systems using the chemically treated Micrococcus luteus IAM 1056 cells (hot water-treated, diluted NaOH-treated, chloroform–methanol-treated, and chloroform–methanol/concentrated KOH-treated cells) was examined. The intact cells of M. luteus adsorbed 527 μmol of copper per g cells, and its copper adsorption was very rapid and was affected by the solution pH. The chloroform–methanol/concentrated KOH-treated cells showed higher copper biosorption capacity than the intact and the other chemically treated cells. The electron paramagnetic resonance (EPR) parameters, g ∥ and |A ∥|, of Cu(II) ion in microbial cells indicate that Cu(II) ion in the intact and all the chemically treated cells have coordination environments with nitrogen and oxygen as donor atoms, being similar to those of type II proteins. The parameter g ∥ also indicated that the coupling between Cu(II) ion and the cell materials in the CHCl3–MeOH/concentrated KOH-treated cells is rather more stable than those between Cu(II) ion and the cell materials in the other treated cells.

Antibacterial Activity of Bacillus amyloliquefaciens Phage Endolysin without Holin Conjugation.

To characterize the enzymatic activity and antibacterial activity of endolysin encoded by a Bacillus amyloliquefaciens phage, the open reading frame encoding endolysin was amplified by PCR and cloned into the expression plasmid pET21d(+). The resultant plasmid was used to transform Escherichia coli JM109(DE3). Production of endolysin in the cytosol facilitated cell lysis without coproduction of holin, which is considered to degrade or alter the cytoplasmic membrane. The phage endolysin was overexpressed and purified. Although the specific activity of the purified phage endolysin towards lyophilized Micrococcus luteus cells was 1/11 of the activity of chicken egg white lysozymes, the endolysin showed stronger antibacterial activity towards E. coli W3110, E. coli JM109(DE3) and Pseudomonas aeruginosa PAO1 than chicken egg white lysozymes. The antibacterial activity of the endolysin towards these three bacterial strains was marked when EDTA was added to the endolysin solution.

Some properties of a macromolecular conjugate of lysozyme prepared by modification with a monomethoxypolyethylene glycol derivative.

Hen egg-white lysozyme was modified with a succinyl ester derivative of monomethoxypolyethylene glycol (mPEG-COONSu), and some properties of the resulting conjugate (mPEG-lysozyme) were studied. The conjugate was prepared by modification of lysozyme with mPEG-COONSu and purified with use of columns of CM-Toyopearl 650M and Sephadex G-75. Analytical data indicated that in the conjugate, 1.05 moles of mPEG with an average molecular weight of 5,000 were covalently attached to the lysozyme molecule. Tryptic peptide analysis of the conjugate showed that Lys 33 in lysozyme is the residue mainly modified with mPEG-COONSu. Covalent attachment of the mPEG-derivative to amino groups greatly increased the thermostability of lysozyme without any conformational change of the protein molecule. mPEG-lysozyme retained full enzyme activity for glycol chitin, but lytic activity for Micrococcus luteus cells in neutral media was 75% of that of native lysozyme and its optimal pH was at pH 5.0. It was also found that the reactivity of lysozyme with anti-lysozyme antibody from BALB/c mice or human lymphocytes was decreased by modification with mPEG-COONSu. From these findings, it was suggested that mPEG-COONSu can be advantageously used for protein tailoring of lysozyme.

Protein purification and gene isolation of chlamysin, a cold-active lysozyme-like enzyme with antibacterial activity

An antibacterial ∼11 kDa protein designated chlamysin was isolated from viscera of the marine bivalve Chlamys islandica. Chlamysin inhibited the growth of all Gram-positive and Gram-negative bacteria tested. The isolated protein was highly efficient in hydrolyzing Micrococcus luteus cells only at low pH (4.5–6.2) and at low temperature (4–35°C). No significant loss of enzyme activity was observed after 30 days storage at room temperature or after heating to 70°C for 15 min, suggesting relatively high protein structure stability. Sequence-analyzed fragments of the protein revealed data which guided the isolation of the cDNA gene, encoding a 137 amino acid chlamysin precursor in scallops. The deduced protein contains a high portion of cysteine, serine and histidine residues and has a predicted isoelectric point below 7. The chlamysin protein was found to have sequence homology to an isopeptidase and to a recently published bivalve lysozyme.

Physiological characterization of Streptococcus bovis mutants that can resist 2-deoxyglucose-induced lysis.

Streptococcus bovis JB1 does not normally lyse, but stationary phase lysis can be induced by including 2-deoxyglucose (2DG) in the growth medium. Isolates deficient in glucose/2DG phosphotransferase activity (PTS-) also lysed when 2DG was present (Lys+) and this result indicated that 2DG phosphorylation via the PTS was not an obligate requirement for 2DG-induced lysis. Cells and cell walls from 2DG-grown cultures lysed faster when proteinase K was added, but glucose-grown cultures and cell walls were not affected. A lipoteichoic acid (LTA) extract (aqueous phase from hot phenol treatment) from glucose-grown cells inhibited the lysis of 2DG-grown cultures, but a similar extract prepared from 2DG-grown cells was without effect. Thin-layer chromatography and differential staining indicated that wild-type and Lys+ PTS- cells incorporated 2DG into LTA, but lysis-resistant cultures (Lys- PTS+ and Lys- PTS-) did not. LTA from lysis-resistant (Lys- PTS+ and Lys- PTS-) cells grown with glucose and 2DG also prevented 2DG-dependent lysis of the wild-type. LTA could not inhibit degradation of cell walls isolated from 2DG-grown cultures, but LTA inhibited the lysis of Micrococcus lysodeikticus (Micrococcus luteus) cells that were exposed to supernatants from 2DG-grown S. bovis cultures. Group D streptococci (including S. bovis) normally have an alpha-1,2 linked glucose disaccharide (kojibiose) in their LTA, but kojibiose cannot be synthesized from 2DG. This observation suggested that the kojibiose moiety of LTA was involved in autolysin inactivation. Wild-type S. bovis had ATP- as well as PEP-dependent mechanisms of 2DG phosphorylation and one lysis-resistant phenotype (Lys- PTS-) had reduced levels of both activities. However, the Lys- PTS+ phenotype was still able to phosphorylate 2DG via ATP and PEP and this result indicated that some other step of 2DG incorporation into LTA was being inhibited. Based on these results, growth in the presence of 2DG appears to prevent synthesis of normal LTA, which is involved in the regulation of autolytic enzymes.

Isolation and purification of a Campylobacter upsaliensis autolysin.

Autolytic activity in the soluble and sediment fractions of sonicates of the spiral and the coccoid form of Campylobacter upsaliensis could not be demonstrated by native (nondenaturing) polyacrylamide gel electrophoresis (PAGE). Autolysins were detected, however, by using denaturing sodium dodecyl sulfate (SDS)-PAGE gels containing either purified Escherichia coli peptidoglycan or whole cells of Micrococcus luteus (Micrococcus lysodeikticus) as the turbid substrate, with subsequent renaturation by treatment with Triton X-100 buffer. In renaturing gels that contained Escherichia coli peptidoglycan, 14 putative autolytic bands ranging from 200 to 12 kDa were detected. In similar gels containing whole cells of M. luteus, only a single band appeared with a molecular mass of 34 kDa. This band corresponded to one of the bands present in the gels containing Escherichia coli peptidoglycan. This common autolysin was isolated by adsorbing it from Campylobacter upsaliensis soluble fractions onto M. luteus cells and then subjecting these cells to renaturing SDS-PAGE in gels containing Escherichia coli peptidoglycan. The 34-kDa autolysin differed from a single 51-kDa autolysin unique to the M. luteus cells, and when isolated from an SDS-PAGE gel, was pure when tested by isoelectric focusing. The N-terminal amino acid sequence analysis showed the first 15 amino acids of the 34-kDa autolysin to have 67% identity to a part of antigenic protein PEB4 of Campylobacter jejuni. The purified autolysin was used to immunize rabbits and the antibodies produced precipitated autolytic activity from cell lysates. The specificity of the antibodies was shown by Western blotting: only a single specific band occurred, with a molecular mass of 34 kDa, and thus it seems unlikely that the 34-kDa autolysin was derived from any of the other autolysins that were detected.

Isolation and purification of a Campylobacter upsaliensis autolysin

Autolytic activity in the soluble and sediment fractions of sonicates of the spiral and the coccoid form of Campylobacter upsaliensis could not be demonstrated by native (nondenaturing) polyacrylamide gel electrophoresis (PAGE). Autolysins were detected, however, by using denaturing sodium dodecyl sulfate (SDS) - PAGE gels containing either purified Escherichia coli peptidoglycan or whole cells of Micrococcus luteus (Micrococcus lysodeikticus) as the turbid substrate, with subsequent renaturation by treatment with Triton X-100 buffer. In renaturing gels that contained Escherichia coli peptidoglycan, 14 putative autolytic bands ranging from 200 to 12 kDa were detected. In similar gels containing whole cells of M. luteus, only a single band appeared with a molecular mass of 34 kDa. This band corresponded to one of the bands present in the gels containing Escherichia coli peptidoglycan. This common autolysin was isolated by adsorbing it from Campylobacter upsaliensis soluble fractions onto M. luteus cells and then subjecting these cells to renaturing SDS-PAGE in gels containing Escherichia coli peptidoglycan. The 34-kDa autolysin differed from a single 51-kDa autolysin unique to the M. luteus cells, and when isolated from an SDS-PAGE gel, was pure when tested by isoelectric focusing. The N-terminal amino acid sequence analysis showed the first 15 amino acids of the 34-kDa autolysin to have 67% identity to a part of antigenic protein PEB4 of Campylobacter jejuni. The purified autolysin was used to immunize rabbits and the antibodies produced precipitated autolytic activity from cell lysates. The specificity of the antibodies was shown by Western blotting: only a single specific band occurred, with a molecular mass of 34 kDa, and thus it seems unlikely that the 34-kDa autolysin was derived from any of the other autolysins that were detected.Key words: autolysin, Campylobacter upsaliensis, zymogram, murein hydrolase.

On resuscitation from the dormant state of Micrococcus luteus.

It has been found previously that a significant number of Micrococcus luteus cells starved in a prolonged stationary phase (up to 2 months) and then held on the bench at room temperature without agitation for periods of up to a further 2-7 months can be resuscitated in liquid media which contained (statistically) no initially-viable (colony-forming) cells but which were fortified with sterile supernatant from the late logarithmic phase of batch growth. Here it was found that such resuscitation can be done only within a defined time period after taking the first sample from such cultures, necessarily involving agitation of the cells. The duration of this period depends on the age of the starved culture: cells kept on the bench for 3 months possess a 2 month period of resuscitability while cells starved for 6 months can be resuscitated only within 10 days after the beginning of sampling. It is suggested that the input of oxygen to the starved cultures while they are agitated may exert a negative influence on the cells, since cultures stored in anaerobic conditions (under nitrogen) had a more prolonged 'survival' time. The cells which experienced between 10 and 60 days of starvation on the bench could be resuscitated, although the number of resuscitable cells depended strongly on the concentration of yeast extract in the resuscitation medium. This concentration for cells stored on the bench for more than 2 months was 0.05% while '1-month-old' cells displayed a maximum resuscitability in the presence of 0.01% of yeast extract. Application of the fluorescent probe propidium iodide revealed the formation of cells with a damaged permeability barrier if resuscitation was performed by using concentrations of yeast extract of 0.1% and above. Thus the successful resuscitation of bacterial cultures under laboratory conditions may need rather strictly defined parameters if it is to be successfully performed for the majority of cells in a population.

Importance of van der Waals contact between Glu 35 and Trp 109 to the catalytic action of human lysozyme

The importance of van der Waals contact between Glu 35 and Trp 109 to the active-site structure and the catalytic properties of human lysozyme (HL) has been investigated by site-directed mutagenesis. The X-ray analysis of mutant HLs revealed that both the replacement of Glu 35 by Asp or Ala, and the replacement of Trp 109 by Phe or Ala resulted in a significant but localized change in the active-site cleft geometry. A prominent movement of the backbone structure was detected in the region of residues 110 to 120 and in the region of residues 100 to 115 for the mutations concerning Glu 35 and Trp 109, respectively. Accompanied by the displacement of the main-chain atoms with a maximal deviation of C alpha atom position ranging from 0.7 A to 1.0 A, the mutant HLs showed a remarkable change in the catalytic properties against Micrococcus luteus cell substrate as compared with native HL. Although the replacement of Glu 35 by Ala completely abolished the lytic activity, HL-Asp 35 mutant retained a weak but a certain lytic activity, showing the possible involvement of the side-chain carboxylate group of Asp 35 in the catalytic action. The kinetic consequence derived from the replacement of Trp 109 by Phe or Ala together with the result of the structural change suggested that the structural detail of the cleft lobe composed of the residues 100 to 115 centered at Ala 108 was responsible for the turnover in the reaction of HL against the bacterial cell wall substrate. The results revealed that the van der Waals contact between Glu 35 and Trp 109 was an essential determinant in the catalytic action of HL.

Micrococcus luteus cells and cell walls induce anaphylactoid reactions accompanied by early death and serum cytokines in mice primed with muramyl dipeptide

Micrococcus luteus strains at a dose of 500 μg of whole cells caused anaphylactoid reactions leading to death in some instances within 1 h in C3H/HeN mice primed with muramyl dipeptide (MDP, 100 μg). Tumor necrosis factor (TNF) and interleukin-6 (IL-6) were induced in the serum of half and of all the surviving mice, respectively. Cell wall specimens of M. luteus so far examined also caused anaphylactoid reactions accompanied by early death and one strain induced high levels of TNF and IL-6. Cytoplasmic membranes also induced IL-6. Essentially similar results were obtained with representative M. luteus cells and a cell wall specimen in MDP-primed C3H/HeJ mice. These results indicate that M. luteus has virulence activities that are associated with the induction of septic shock and systemic inflammatory diseases.

Biosynthesis of Teichuronic Acid in the Bacterial Cell Wall: PURIFICATION AND CHARACTERIZATION OF THE GLUCOSYLTRANSFERASE OF MICROCOCCUS LUTEUS

This report describes what is, to our knowledge, the first purification to near homogeneity of an enzyme involved in the biosynthesis of the teichuronic acid of Micrococcus luteus cell walls. The glucosyltransferase of M. luteus, which participates in the biosynthesis of teichuronic acid, was solubilized from cytoplasmic membrane fragments by extraction with buffer solutions containing the detergents Thesit (dodecyl alcohol polyoxyethylene ether; 1 mg/ml) and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (0.5 mg/ml). The detergent-solubilized enzyme was purified 150-fold, with a recovery of 13% by adsorbent column chromatography, ion-exchange chromatography, gel filtration, and preparative nondenaturing gradient polyacrylamide gel electrophoresis. On the basis of its mobility on native gradient gel, the glucosyltransferase was estimated to have a molecular mass of 440 kDa. The purified native enzyme was a multisubunit protein consisting of subunits of two sizes; their molecular masses were determined to be 52.5 and 54 kDa, respectively, by observation of the mobility of the protein bands in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point of the enzyme was approximately 5.

Quantitative Analysis of the Physiological Heterogeneity within Starved Cultures of Micrococcus luteus by Flow Cytometry and Cell Sorting

A high proportion of Micrococcus luteus cells in cultures which had been starved for 3 to 6 months lost the ability to grow and form colonies on agar plates but could be resuscitated from their dormancy by incubation in an appropriate liquid medium (A. S. Kaprelyants and D. B. Kell, Appl. Environ. Microbiol. 59:3187-3196, 1993). We used flow cytometry and cell sorting to study populations of bacteria that had been starved for 5 months. These cells could be stained by the fluorescent lipophilic cation rhodamine 123, but such staining was almost independent of metabolically generated energy in that it was not affected by uncouplers. Two populations could be distinguished, one with a lower degree of rhodamine fluorescence (a degree of fluorescence referred to as region A and containing approximately 80% of the cells) and one with a more elevated degree of fluorescence (region B, approximately 20% of the cells). Subsequent incubation of starved cells in fresh medium in the presence of the antibiotic chloramphenicol (to which M. luteus is sensitive) resulted in the transient appearance of cells actively accumulating rhodamine 123 (and fluorescing in region B) and of larger cells exhibiting a yet-greater degree of fluorescence (region C). These more fluorescent cells accounted for as much as 50% of the total population, under conditions in which the viable and total counts were constant. Thus, metabolic resuscitation of at least one-half of the cells takes place under conditions in which cryptic growth cannot play any role. Sorting experiments revealed that the great majority of the viable cells in the starved population are concentrated in regions B and C and that the extent of rhodamine staining under conditions of starvation therefore reflects the physiological state of the cells. Physical separation of these cells from cells in region A resulted in an increase (of approximately 25-fold) in the viability of cells in regions B and C and of the population as a whole. Resuscitation of dormant cells in a most-probable-number assay in the presence of supernatant taken from growing M. luteus revealed the resuscitation of cells from regions B and C but not from region A. It is suggested that initially dormant (resuscitable) cells are concentrated in regions B and C.

Synergistic contributions of asparagine 46 and aspartate 52 to the catalytic mechanism of chicken egg white lysozyme.

The X-ray structure of a chicken egg white lysozyme (ChEWL) complex with a peptidoglycan-derived inhibitor suggests that interactions of Asn46 and Asp52 with the D-subsite N-acetylmuramic acid residue help to distort that pyranose ring into the reactive half-chair conformation and that a hydrogen bond is formed between Asn46 and Asp52 [Strynadka, N. C. J., & James, M. N. G. (1991) J. Mol. Biol. 220, 401-424]. These hypotheses were investigated through the D52A, N46A, and D52A/N46A mutants of ChEWL. The Michaelis constants of the D52A and D52A/N46A ChEWL complexes with Micrococcus luteus cells are 3- and 4-fold higher, respectively, than the wild-type KM; the corresponding kcat values are 25- and 50-fold lower, respectively, than the wild-type kcat. These results support the proposal of Strynadka and James. The velocities of reactions catalyzed by the N46A and D52A mutants are approximately equal to each other for all classes of substrate, suggesting that the respective roles of Asn46 and Asp52 in transition state stabilization do not vary. The mutation of either Asn46 or Asp52 to Ala apparently disrupts the interactions of the other (nonmutated) residue with the substrate, supporting the crystallographic evidence of a hydrogen-bond interaction between the two residues. The mutations do not change the values of the dissociation constants of complexes with (carboxymethyl)chitin complexes, suggesting that ground state complexes of ChEWL with chitin-derived substrates differ in conformation from complexes with bacterial peptidoglycans.

Origin of carbohydrate recognition specificity of human lysozyme revealed by affinity labeling.

In order to reveal the origin of carbohydrate recognition specificity of human lysozyme by clarifying the difference in the binding mode of ligands in the active site, the inactivation of human lysozyme by 2',3'-epoxypropyl beta-glycoside derivatives of the disaccharides, N,N'-diacetylchitobiose [GlcNAc-beta-(1-->4)-GlcNAc] and N-acetyllactosamine [Gal-beta-(1-->4)-GlcNAc], was investigated and the three-dimensional structures of the affinity-labeled enzymes were determined by X-ray crystallography at 1.7 A resolution. Under the conditions comprising 2.0 x 10(-3) M labeling reagent and 1.0 x 10(-5) M human lysozyme at pH 5.4, 37 degrees C, the reaction time required to reduce the lytic activity against Micrococcus luteus cells to 50% of its initial activity was lengthened by 3.7 times through the substitution of the nonreducing end sugar residue, GlcNAc to Gal. The refined structure of human lysozyme labeled by 2',3'-epoxypropyl beta-glycoside derivatives of N,N'-diacetylchitobiose (HL/NAG-NAG-EPO complex) indicated that the interaction mode of the N,N'-diacetylchitobiose moiety in substites B and C in this study was essentially the same as in the case of the complex of human lysozyme with the free ligand. On the other hand, the hydrogen-bonding pattern and the stacking interaction at subsite B were remarkably different between the HL/NAG-NAG-EPO complex and human lysozyme labeled by the 2',3'-epoxypropyl beta-glycoside of N-acetyllactosamine (HL/GAL-NAG-EPO complex). The reduced number of possible hydrogen bonds as well as the less favorable stacking between the side chain of Tyr63 in human lysozyme and the galactose residue in the HL/GAL-NAG-EPO complex reasonably explained the less efficient ability of the 2',3'-epoxypropyl beta-glycoside of N-acetyllactosamine as compared to that of N,N'-diacetylchitobiose as an affinity labeling reagent toward human lysozyme.