Design of an adenosine phosphorylase by active-site modification of murine purine nucleoside phosphorylase. Enzyme kinetics and molecular dynamics simulation of Asn-243 and Lys-244 substitutions of purine nucleoside phosphorylase.

Our objective was to alter the substrate specificity of purine nucleoside phosphorylase such that it would catalyse the phosphorolysis of 6-aminopurine nucleosides. We modified both Asn-243 and Lys-244 in order to promote the acceptance of the C6-amino group of adenosine. The Asn-243-Asp substitution resulted in an 8-fold increase in K(m) for inosine from 58 to 484 microM and a 1000-fold decrease in k(cat)/K(m). The Asn-243-Asp construct catalysed the phosphorolysis of adenosine with a K(m) of 45 microM and a k(cat)/K(m) 8-fold that with inosine. The Lys-244-Gln construct showed only marginal reduction in k(cat)/K(m), 83% of wild type, but had no activity with adenosine. The Asn-243-Asp;Lys-244-Gln construct had a 14-fold increase in K(m) with inosine and 7-fold decrease in k(cat)/K(m) as compared to wild type. This double substitution catalysed the phosphorolysis of adenosine with a K(m) of 42 microM and a k(cat)/K(m) twice that of the single Asn-243-Asp substitution. Molecular dynamics simulation of the engineered proteins with adenine as substrate revealed favourable hydrogen bond distances between N7 of the purine ring and the Asp-243 carboxylate at 2.93 and 2.88 A, for Asn-243-Asp and the Asn-243-Asp;Lys-244-Gln constructs respectively. Simulation also supported a favourable hydrogen bond distance between the purine C6-amino group and Asp-243 at 2.83 and 2.88 A for each construct respectively. The Asn-243-Thr substitution did not yield activity with adenosine and simulation gave unfavourable hydrogen bond distances between Thr-243 and both the C6-amino group and N7 of the purine ring. The substitutions were not in the region of phosphate binding and the apparent S(0.5) for phosphate with wild type and the Asn-243-Asp enzymes were 1.35+/-0.01 and 1.84+/-0.06 mM, respectively. Both proteins exhibited positive co-operativity with phosphate giving Hill coefficients of 7.9 and 3.8 respectively.

Physical and functional S-layer reconstitution in Aeromonas salmonicida.

The various functions attributed to the S-layer of Aeromonas salmonicida have been previously identified by their conspicuous absence in S-layer-defective mutants. As a different approach to establish the multifunctional nature of this S-layer, we established methods for reconstitution of the S-layer of A. salmonicida. Then we investigated the functional competence of the reconstituted S-layer. S-layers were reconstituted in different systems: on inert membranes or immobilized lipopolysaccharide (LPS) from purified S-layer protein (A-protein) or on viable cells from either A-protein or preassembled S-layer sheets. In the absence of divalent cations and LPS, purified A-protein in solution spontaneously assembled into tetrameric oligomers and, upon concentration by ultrafiltration, into macroscopic, semicrystalline sheets formed by oligomers loosely organized in a tetragonal arrangement. In the presence of Ca2+, purified A-protein assembled into normal tetragonal arrays of interlocked subunits. A-protein bound with high affinity (Kd, 1.55 x 10(-7) M) and specificity to high-molecular-weight LPS from A. salmonicida but not to the LPSs of several other bacterial species. In vivo, A-protein could be reconstituted only on A. salmonicida cells which contained LPS, and Ca2+ affected both a regular tetragonal organization of the reattached A-protein and an enhanced reattachment of the A-protein to the cell surface. The reconstitution of preformed S-layer sheets (produced by an S-layer-secreting mutant) to an S-layer-negative mutant occurred consistently and efficiently when the two mutant strains were cocultured on calcium-replete solid media. Reattached A-protein (exposed on the surface of S-layer-negative mutants) was able to bind porphyrins and an S-layer-specific phage but largely lacked regular organization, as judged by its inability to bind immunoglobulins. Reattached S-layer sheets were regularly organized and imparted the properties of porphyrin binding, hydrophobicity, autoaggregation, adherence to and invasion of fish macrophages and epithelial cells, and resistance to macrophage cytotoxicity. However, cells with reconstituted S-layers were still sensitive to complement and insensitive to the antibiotics streptonigrin and chloramphenicol, indicating incomplete functional reconstitution.

Physiological consequences of the S-layer of Aeromonas salmonicida in relation to growth, temperature, and outer membrane permeation.

S-layers are paracrystalline protein multimers that cover the entire cell surface of many bacterial species. The presence of an S-layer in Aeromonas salmonicida (also known as A-layer) predisposed this bacterium to apparently unrelated physiological consequences: inhibition of growth at 30 degrees C, enhanced cell filamentation at 37 degrees C, and enhanced uptake of the hydrophobic antibiotics streptonigrin and chloramphenicol. Growth inhibition or enhanced filamentation was not observed when the native A-layer was missing or its arrangement altered, as in Ca(2+)-limited or Ca(2+)- and Mg(2+)-limited cells, in A-layer-negative (A-) cells with an artificially reconstituted A-layer, or in mutants unable to correctly assemble this layer. A-layer-positive cells (A+) were far more sensitive to the intracellularly acting antibiotics streptonigrin and chloramphenicol than were A- cells, and streptonigrin-resistant mutants were predominantly A-. Hemin, a compound known to specifically bind to the A-layer, alleviated streptonigrin toxicity to A+, but not A-, cells. As well, Ca(2+)- and Mg(2+)-limited cells, or mutants harboring A-layer defects had a reduced sensitivity to streptonigrin, and A- cells with reconstituted A-layers remained resistant to streptonigrin and chloramphenicol. Thus, the presence of a native A-layer arrangement on the cell surface, and not the mere presence of A-layer protein subunits, predisposed A. salmonicida toward the aforementioned physiological consequences. The A-layer is suggested to specifically effect these consequences, in particular the permeation of streptonigrin or chloramphenicol, by a specific interaction of A-layer subunits with the outer membrane.

Novel structural patterns in divalent cation-depleted surface layers of Aeromonas salmonicida.

The fish pathogen Aeromonas salmonicida possesses a regular surface layer (or A-layer) which is an important virulence determinant. The A-protein, a single bilobed protein organized in a p4 lattice of M4C4 arrangement with two morphological domains, comprises this layer. The role of divalent cations in the A-layer structure was studied to better understand A-protein subunit interactions affecting structural flexibility and function. Divalent cation bridges were found to be involved in the integrity of the A-layer. Two novel A-layer patterns were formed as the result of growth under calcium limitation or by chelation of divalent cations with EDTA or EGTA, thereby constituting the first reported case of formation of distinct regular arrays upon divalent cation depletion. Furthermore, under these conditions A-protein was sometimes released as tetrameric units, rather than in monomeric form. The formation of the two novel patterns is best explained by a sequence of structural rearrangements, following disruption of only one of the two A-layer morphological units, that is, those held together by divalent cation bridges. The free tetrameric units represent four A-protein subunits clustered around the unaffected four-fold axis.

Structure of the tetragonal surface virulence array protein and gene of Aeromonas salmonicida.

The paracrystalline surface protein array of the pathogenic bacterium Aeromonas salmonicida is a primary virulence factor with novel binding capabilities. The species-specific structural gene (vapA) for this array protein (A-protein) was cloned into lambda gt11 but was unstable when expressed in Escherichia coli, undergoing an 816-base pair deletion due to a 21-base pair direct repeat within the gene. However, the gene was stable in cosmid pLA2917 as long as expression was poor. A-protein was located in the cytoplasmic, inner membrane and periplasmic fractions in E. coli. The DNA sequence revealed a 1,506-base pair open reading frame encoding a protein consisting of a 21-amino acid signal peptide, and a 481-residue 50,778 molecular weight protein containing considerable secondary structure. When assembled into a paracrystalline protein array on Aeromonas the cell surface A-protein was totally refractile to cleavage by trypsin, but became trypsin sensitive when disassembled. Trypsin cleavage of the isolated protein provided evidence that both the NH2- and COOH-terminal regions form distinct structural domains, consistent with three-dimensional ultrastructural evidence. The NH2-terminal 274-residue domain remained refractile to trypsin activity. This segment connects by a trypsin and CNBr-sensitive 78-residue linker region to a COOH-terminal 129-residue fragment which could apparently refold into a partially trypsin-resistant structure after cleavage at residue 323.

A novel ATPase complex selectively accumulated upon heat shock is a major cellular component of thermophilic archaebacteria.

We have discovered a large cylindrical protein complex which is an abundant component of the cytoplasm of extremely thermophilic archaebacteria. Structural analysis by image processing of electron micrographs suggests that the complex is composed of two stacked rings of eight subunits each; the rings enclose a central channel. The complex purified from the hyperthermophile Pyrodictium occultum is composed of equal quantities of two polypeptides of Mr 56,000 and 59,000. It exhibits an extremely thermostable ATPase activity with a temperature optimum of 100 degrees C. The basal level of the ATPase complex in the cell is high, and it becomes highly enriched as a result of heat shock (shift from 102 degrees C to 108 degrees C) or balanced growth at temperatures near the physiological upper limit. Immunoblotting results indicate that a related protein is present in most thermophilic archaebacteria and in Escherichia coli. This protein complex may play an important role in the adaptation of thermophilic archaebacteria to life at high temperature.

The cell envelope of the hyperthermophilic archaebacterium Pyrobaculum organotrphum consists of two regularly arrayed protein layers: three-dimensional structure of the outer layer.

The cell envelope of the hyperthermophilic sulphur-reducing archaebacterium Pyrobaculum organotrophum H10 was found to be composed of two distinct hexagonally arranged crystalline protein arrays. Electron microscopic analysis of freeze-etched cells and isolated envelopes in conjunction with image processing showed that the inner layer (lattice centre-to-centre spacing 27.9 nm) is essentially identical to the protein array of Pyrobaculum islandicum GEO3, a complex, rigid structure implicated in the maintenance of cell shape. The outer layer has clear p6 symmetry and a lattice spacing of 20.6 nm. Its three-dimensional structure was reconstructed from a negative stain tilt series of an intact double-layered envelope using Fourier filtration to separate the desired information from the other lattices present. The outer layer is a unique, porous network of blocklike dimers disposed around six-fold axes, and exhibits minimal asymmetry between its inner and outer faces. It appears to be rather loosely associated with the outer surface of the inner layer. In most H10 envelopes, the inner layer is orientated with one base vector exactly perpendicular to the long axis of the cell, so that the cylindrical portion is composed of a series of parallel cell-girdling hoops of hexameric morphological units. All the other known Pyrobaculum strains were found to have a GEO3-type envelope structure, consisting of a single rigid protein array and a fibrous capsule. Although H10 does not possess a capsule, fibrils appear to be sandwiched between the two protein layers.

Immunoglobulin binding by the regular surface array of Aeromonas salmonicida.

The cell surface of Aeromonas salmonicida is covered by a regular surface array composed of a single species of protein, the A-protein (Phipps, B. M., Trust, T. J., Ishiguro, E. E., and Kay, W. W. (1983) Biochemistry 22, 2934-2939). The array, known as the A-layer, is the key virulence factor for this organism. Cells containing the A-layer specifically bound rabbit IgG and human IgM with high affinity (KD = 1.0 X 10(-6) M and 3.3 X 10(-6) M, respectively), but neither isogenic A-protein-deficient strains nor an Aeromonas hydrophila strain also possessing a regular surface array had binding activity. Selective removal of A-protein at pH 2.2 inactivated IgG binding. Structurally intact IgG was requisite for binding since both Fab and Fc fragments were inactive. Aeromonas A-protein did not share the same IgG binding sites as Staphylococcus aureus protein A. Purified A-protein bound IgG only weakly, but reassembled A-layer regained binding activity. Protein modification and perturbation of the A-layer indicated that no single amino acid residue was critical for binding, and that the binding site consisted of a native arrangement of at least four A-protein monomers in the layer.

Porphyrin binding by the surface array virulence protein of Aeromonas salmonicida.

Congo red binding by virulent A-layer-containing (A+) and avirulent A-layer-deficient (A-) strains of Aeromonas salmonicida was examined. Congo red binding to A+ cells was enhanced by salt and thus hydrophobically driven, but at low Congo red concentrations binding was salt independent. Congo red was bound by A+ cells by a kinetically distinct mechanism (Kd, 0.25 microM) which was absent in A- isogenic strains. Purified A-layer protein ("A protein") protein A also bound Congo red with similar affinity (Kd, 0.40 microM). Congo red binding was structurally specific; it was not influenced by a wide variety of compounds including amino acids and nucleotides and only weakly inhibited by structurally similar dyes. However, protoporphyrin IX and hemin were strong competitive inhibitors of Congo red binding. Protoporphyrin and hemin were bound only by A+ strains (KdS of 0.41 and 0.63 microM, respectively). Furthermore, binding of these porphyrins was strongly inhibited by Congo red but weakly inhibited by hematoporphyrin. Purified A protein also bound protoporphyrin IX and hemin with affinities similar to those of A+ cells (KdS of 0.94 and 0.41 microM, respectively.

Surface layer virulence A-proteins from Aeromonas salmonicida strains.

Superficial surface layer proteins (A-proteins) were present on diverse isolates of Aeromonas salmonicida which differed both physiologically and in pathogenesis. Three of these proteins were purified directly from the surface of whole cells or from outer membrane preparations. These A-proteins were unusually hydrophobic (45-47%) and of similar but not identical molecular mass (49, 50, and 51 kdaltons). They were nearly identical in amino acid composition and were highly conserved, but not identical with respect to their hydrophobic N-terminal amino acid sequences. These proteins differed, however, with respect to their oligomerization properties, isoelectric forms, and chymotryptic peptide patterns. All three proteins were immunologically closely related and shared surface-exposed immunoreactive peptides with 28 separate isolates.

Purification and characterization of the cell surface virulent A protein from Aeromonas salmonicida.

The predominant cell surface protein (A protein) of Aeromonas salmonicida has been purified in high yield from outer membranes by using a combination of detergent and chaotropic extraction methods as well as exclusion and ion-exchange chromatography. The A protein was primarily monomeric, Mr 50 000, but readily formed oligomers at high protein or low salt concentrations. Several isoelectric forms were distinguishable with purified protein as well as in situ on the cell surface. Neither phosphate nor carbohydrate was detectable. The A protein was hydrophobic in composition and the N-terminal sequence highly hydrophobic. From CD spectra the A protein exhibited 14% alpha helix and 19-28% beta structure and could also be readily crystallized. By fluorescent antibody staining the A protein was shown to cover the entire cell surface but was absent from A protein deficient mutants. This protein appears to have no apparent enzymatic activity but rather constitutes a macromolecular refractile protein barrier essential for virulence.

Purification and disposition of a surface protein associated with virulence of Aeromonas salmonicida.

Virulent strains of Aeromonas salmonicida observed by electron microscopy were characterized by an outer layer exhibiting a tetragonal repeat pattern. Attenuated strains had a 2.5 X 10(3)- to 5 X 10(3)-fold reduction in virulence and lost the outer layer, autoaggregating properties, and a 49-kilodalton protein (A protein) simultaneously. The A protein is the major protein component of outer membrane fractions of virulent strains. A variety of radiolabeling studies showed that this protein was surface localized and that it provided an effective barrier against iodination of other outer membrane proteins with either lactoperoxidase or diazoiodosulfanilic acid; A protein was not labeled with lactoperoxidase but was specifically labeled with diazoidosulfanilic acid. The A protein was purified by selective extraction with detergent and guanidine hydrochloride, and its amino acid composition was determined. The properties of A protein are compared with those of other bacterial surface layer proteins.