Conserved tyrosine-369 in the active site of Escherichia coli copper amine oxidase is not essential.

Copper amine oxidases are homodimeric enzymes that catalyze two reactions: first, a self-processing reaction to generate the 2,4,5-trihydroxyphenylalanine (TPQ) cofactor from an active site tyrosine by a single turnover mechanism; second, the oxidative deamination of primary amine substrates with the production of aldehyde, hydrogen peroxide, and ammonia catalyzed by the mature enzyme. The importance of active site residues in both of these processes has been investigated by structural studies and site-directed mutagenesis in enzymes from various organisms. One conserved residue is a tyrosine, Tyr369 in the Escherichia coli enzyme, whose hydroxyl is hydrogen bonded to the O4 of TPQ. To explore the importance of this site, we have studied a mutant enzyme in which Tyr369 has been mutated to a phenylalanine. We have determined the X-ray crystal structure of this variant enzyme to 2.1 A resolution, which reveals that TPQ adopts a predominant nonproductive conformation in the resting enzyme. Reaction of the enzyme with the irreversible inhibitor 2-hydrazinopyridine (2-HP) reveals differences in the reactivity of Y369F compared with wild type with more efficient formation of an adduct (lambda(max) = 525 nm) perhaps reflecting increased mobility of the TPQ adduct within the active site of Y369F. Titration with 2-HP also reveals that both wild type and Y369F contain one TPQ per monomer, indicating that Tyr369 is not essential for TPQ formation, although we have not measured the rate of TPQ biogenesis. The UV-vis spectrum of the Y369F protein shows a broader peak and red-shifted lambda(max) at 496 nm compared with wild type (480 nm), consistent with an altered electronic structure of TPQ. Steady-state kinetic measurements reveal that Y369F has decreased catalytic activity particularly below pH 6.5 while the K(M) for substrate beta-phenethylamine increases significantly, apparently due to an elevated pK(a) (5.75-6.5) for the catalytic base, Asp383, that should be deprotonated for efficient binding of protonated substrate. At pH 7.0, the K(M) for wild type and Y369F are similar at 1.2 and 1.5 microM, respectively, while k(cat) is decreased from 15 s(-1) in wild type to 0.38 s(-1), resulting in a 50-fold decrease in k(cat)/K(M) for Y369F. Transient kinetics experiments indicate that while the initial stages of enzyme reduction are slower in the variant, these do not represent the rate-limiting step. Previous structural and solution studies have implicated Tyr369 as a component of a proton shuttle from TPQ to dioxygen. The moderate changes in kinetic parameters observed for the Y369F variant indicate that if this is the case, then the absence of the Tyr369 hydroxyl can be compensated for efficiently within the active site.

Identification of critical residues in the active site of porcine membrane-bound aminopeptidase P.

The membrane-bound form of mammalian aminopeptidase P (AP-P; EC 3.4. 11.9) is a mono-zinc-containing enzyme that lacks any of the typical metal binding motifs found in other zinc metalloproteases. To identify residues involved in metal binding and catalysis, sequence and structural information was used to align the sequence of porcine membrane-bound AP-P with other members of the peptidase clan MG, including Escherichia coli AP-P and methionyl aminopeptidases. Residues predicted to be critical for activity were mutated and the resultant proteins were expressed in COS-1 cells. Immunoelectrophoretic blot analysis was used to compare the levels of expression of the mutant proteins, and their ability to hydrolyze bradykinin and Gly-Pro-hydroxyPro was assessed. Asp449, Asp460, His523, Glu554, and Glu568 are predicted to serve as metal ion ligands in the active site, and mutagenesis of these residues resulted in fully glycosylated proteins that were catalytically inactive. Mutation of His429 and His532 also resulted in catalytically inactive proteins, and these residues, by analogy with E. coli AP-P, are likely to play a role in shuttling protons during catalysis. These studies indicate that mammalian membrane-bound AP-P has an active-site configuration similar to that of other members of the peptidase clan MG, which is compatible with either a dual metal ion model or a single metal ion in the active site. The latter model is consistent, however, with the known metal stoichiometry of both the membrane-bound and cytosolic forms of AP-P and with a recently proposed model for methionyl aminopeptidase.

Crystal structure of intact elongation factor EF-Tu from Escherichia coli in GDP conformation at 2.05 A resolution.

The crystal structure of intact elongation factor Tu (EF-Tu) from Escherichia coli in GDP-bound conformation has been determined using a combination of multiple isomorphous replacement (MIR) and multiwavelength anomalous diffraction (MAD) methods. The current atomic model has been refined to a crystallographic R factor of 20.3 % and free R-factor of 26.8 % in the resolution range of 10-2.05 A. The protein consists of three domains: domain 1 has an alpha/beta structure; while domain 2 and domain 3 are beta-barrel structures. Although the global fold of the current model is similar to those of published structures, the secondary structural assignment has been improved due to the high quality of the current model. The switch I region (residues 40-62) is well ordered in this structure. Comparison with the structure of EF-Tu in GDP-bound form from Thermus aquaticus shows that although the individual domain structures are similar in these two structures, the orientation of domains changes significantly. Interactions between domains 1 and 3 in our E. coli EF-Tu-GDP complex are quite different from those of EF-Tu with bound GTP from T. aquaticus, due to the domain rearrangement upon GTP binding. The binding sites of the Mg2+ and guanine nucleotide are revealed in detail. Two water molecules that co-ordinate the Mg2+ have been identified to be well conserved in the GDP and GTP-bound forms of EF-Tu structures, as well as in the structure of Ras p21 with bound GDP. Comparisons of the Mg2+ binding site with other guanine nucleotide binding proteins in GDP-bound forms show that the Mg2+ co-ordination patterns are well preserved among these structures.

Catalytic mechanism of the quinoenzyme amine oxidase from Escherichia coli: exploring the reductive half-reaction.

The crystal structure of the complex between the copper amine oxidase from Escherichia coli (ECAO) and a covalently bound inhibitor, 2-hydrazinopyridine, has been determined to a resolution of 2.0 A. The inhibitor covalently binds at the 5 position of the quinone ring of the cofactor, 2,4,5-trihydroxyphenylalaninequinone (TPQ). The inhibitor complex is analogous to the substrate Schiff base formed during the reaction with natural monoamine substrate. A proton is abstracted from a methylene group adjacent to the amine group by a catalytic base during the reaction. The inhibitor, however, has a nitrogen at this position, preventing proton abstraction and trapping the enzyme in a covalent complex. The electron density shows this nitrogen is hydrogen bonded to the side chain of Asp383, a totally conserved residue, identifying it as the probable catalytic base. The positioning of Asp383 is such that the pro-S proton of a substrate would be abstracted, consistent with the stereospecificity of the enzyme determined by 1H NMR spectroscopy. Site-directed mutagenesis and in vivo suppression have been used to substitute Asp383 for 12 other residues. The resulting proteins either lack or, in the case of glutamic acid, have very low enzyme activity consistent with an essential catalytic role for Asp383. The O4 position on the quinone ring is involved in a short hydrogen bond with the hydroxyl of conserved residue Tyr369. The distance between the oxygens is less than 2.5 A, consistent with a shared proton, and suggesting ionization at the O4 position of the quinone ring. The Tyr369 residue appears to play an important role in stabilizing the position of the quinone/inhibitor complex. The O2 position on the quinone ring is hydrogen bonded to the apical water ligand of the copper. The basal water ligand, which lies 2.0 A from the copper in the native structure, is at a distance of 3.0 A in the complex. In the native structure, the active site is completely buried, with no obvious route for entry of substrate. In the complex, the tip of the pyridine ring of the bound inhibitor is on the surface of the protein at the edge of the interface between domains 3 and 4, suggesting this as the entry point for the amine substrate.

Crystallization and preliminary crystallographic analysis of RepA1, a replication control protein of the RepFIC replicon of enterotoxin plasmid EntP307.

RepA1 protein is essential for replication of the RepFIC replicon of enterotoxin plasmid EntP307 and is thought to interact directly with the origin of replication. We have purified RepA1 from an over-producing expression system and have prepared single crystals using a macroseeding technique. The crystals belong to space group P2(1)2(1)2(1) or P2(1)2(1)2, with cell dimensions a = 61 A, b = 67 A, and c = 243 A. They diffract X-rays to 3.3 A resolution and probably contain two 40,000 molecular weight RepA1 molecules per asymmetric unit.

Crystal structure of a quinoenzyme: copper amine oxidase of Escherichia coli at 2 A resolution.

BACKGROUND: Copper amine oxidases are a ubiquitous and novel group of quinoenzymes that catalyze the oxidative deamination of primary amines to the corresponding aldehydes, with concomitant reduction of molecular oxygen to hydrogen peroxide. The enzymes are dimers of identical 70-90 kDa subunits, each of which contains a single copper ion and a covalently bound cofactor formed by the post-translational modification of a tyrosine side chain to 2,4,5-trihydroxyphenylalanine quinone (TPQ). RESULTS: The crystal structure of amine oxidase from Escherichia coli has been determined in both an active and an inactive form. The only structural differences are in the active site, where differences in copper coordination geometry and in the position and interactions of the redox cofactor, TPQ, are observed. Each subunit of the mushroom-shaped dimer comprises four domains: a 440 amino acid C-terminal beta sandwich domain, which contains the active site and provides the dimer interface, and three smaller peripheral alpha/beta domains (D1-D3), each of about 100 amino acids. D2 and D3 show remarkable structural and sequence similarity to each other and are conserved throughout the quinoenzyme family. In contrast, D1 is absent from some amine oxidases. The active sites are well buried from solvent and lie some 35 A apart, connected by a pair of beta hairpin arms. CONCLUSIONS: The crystal structure of E. coli copper amine oxidase reveals a number of unexpected features and provides a basis for investigating the intriguing similarities and differences in catalytic mechanism of members of this enzyme family. In addition to the three conserved histidines that bind the copper, our studies identify a number of other conserved residues close to the active site, including a candidate for the catalytic base and a fourth conserved histidine which is involved in an interesting intersubunit interaction.

Reduced vision secondary to pigmented cellular membranes on silicone intraocular lenses.

PURPOSE: Visually significant, pigmented cellular membranes may form on intraocular lenses after implantation. We studied a series of patients to determine the onset, visual significance, treatment, and recurrent nature of these pigmented membranes in patients who underwent surgery with silicone lens implantation. METHODS: In nine eyes (eight patients) with visually significant pigmented cellular membranes on their Allergan Medical Optics (Irvine, California) silicone intraocular lenses (model SI18NGB or SI26NB), eight underwent combined phacoemulsification, intraocular lens implantation, and trabeculectomy; one underwent phacoemulsification and lens implantation only. We reviewed medical records to identify preoperative, operative, and postoperative similarities and differences in care. Clinical examinations and slit-lamp photographs, over an average of 21.3 +/- 7.2 months (range, 11 to 31 months), documented the effects of different treatment modalities. RESULTS: The patients sought treatment ten to 20 weeks (mean, 15 weeks) postoperatively. Except for the cellular membranes, each eye was without evidence of inflammation or cystoid macular edema. Subjective complaints decreased, and best-corrected Snellen visual acuity improved with topical corticosteroid therapy alone. Pretreatment best-corrected visual acuities ranged from 20/40 to 20/400 (mean, 20/70 using Snellen fractions) and improved two to eight (mean, five) Snellen lines, to a range of 20/20 to 20/50 (mean, 20/25). All nine eyes had recurrence of the membranes after treatment terminated and required a maintenance regimen of corticosteroid eyedrops. CONCLUSION: The pigmented cellular membranes observed on these silicone lenses were visually significant to each patient. The membranes resolved and visual acuity improved with topical corticosteroid treatment alone but recurred in all patients on cessation of treatment.

Crystal structure of a replication fork single-stranded DNA binding protein (T4 gp32) complexed to DNA.

The single-stranded DNA (ssDNA) binding protein gp32 from bacteriophage T4 is essential for T4 DNA replication, recombination and repair. In vivo gp32 binds ssDNA as the replication fork advances and stimulates replisome processivity and accuracy by a factor of several hundred. Gp32 binding affects nearly every major aspect of DNA metabolism. Among its important functions are: (1) configuring ssDNA templates for efficient use by the replisome including DNA polymerase; (2) melting out adventitious secondary structures; (3) protecting exposed ssDNA from nucleases; and (4) facilitating homologous recombination by binding ssDNA during strand displacement. We have determined the crystal structure of the gp32 DNA binding domain complexed to ssDNA at 2.2 A resolution. The ssDNA binding cleft comprises regions from three structural subdomains and includes a positively charged surface that runs parallel to a series of hydrophobic pockets formed by clusters of aromatic side chains. Although only weak electron density is seen for the ssDNA, it indicates that the phosphate backbone contacts an electropositive cleft of the protein, placing the bases in contact with the hydrophobic pockets. The DNA mobility implied by the weak electron density may reflect the role of gp32 as a sequence-independent ssDNA chaperone allowing the largely unstructured ssDNA to slide freely through the cleft.

Structure determination of turkey egg-white lysozyme using Laue diffraction data.

The three-dimensional structure of turkey egg-white lysozyme (TEWL) has been solved and refined at 2.5 A resolution using X-ray data collected by the Laue method. This is the first protein structure determination undertaken using Laue diffraction data. A re-examination of the existing structure of TEWL was necessary when attempts to refine an atomic model based on the C alpha positions in the Protein Data Bank (entry 1LZ2) failed. The correct orientation and position of the turkey lysozyme molecules within the crystallographic unit cell were determined by molecular replacement using a refined model of the homologous hen egg-white lysozyme crystal structure. After modification of the model to reflect the differences in amino-acid sequence between the chicken and turkey enzymes, the structure was subjected to crystallographic refinement using the simulated-annealing refinement technique and conventional least-squares refinement. This yielded a final residual of R = 20.7%. This crystal form is of potential interest for time-resolved crystallographic studies since the amino-acid residues involved in catalysis (Asp52 and Glu35) are accessible to solvent and not blocked by crystal contacts.

Nocardia asteroides keratitis associated with extended-wear soft contact lenses.

Chronic unilateral keratitis developed in a 34-year-old woman who wore extended-wear contact lenses. A clinical response was not obtained until Nocardia asteroides was correctly identified as the causal agent. The response to 30% sulfacetamide was dramatic. We review the clinical presentation of Nocardia keratitis and recommendations for management. Use of corticosteroids should be avoided in Nocardia keratitis. This opportunistic organism should be considered in patients who wear contact lenses in whom infectious keratitis develops.

Posterior chamber intraocular lenses in a series of 75 autopsy eyes. Part II: Postimplantation loop configuration.

We illustrate, from the pathologist's viewpoint, the configuration of posterior chamber lens loops within the eye. The final configuration of these loops is often a "C" shape, particularly following capsular fixation. When one or both loops are placed in the ciliary sulcus, where tissue resistance to loop compressive forces is less than in the capsular sac, an invagination or erosion of the loop into the ciliary body stroma or ciliary muscle frequently occurs. Although a surgeon should first and foremost choose a lens design with which he or she is comfortable in terms of ease of implantation and good clinical results, our pathological observations indicate that a subtle compromise in loop configuration might provide a more physiologically correct configuration in the eye, particularly for those who prefer in-the-bag implantation. Most surgeons prefer a lens design that will provide adequate fixation in the ciliary sulcus if an attempted capsular sac implantation is not successful.

The serology of experimental Salmonella dublin infections of cattle.

Agglutinating antibodies to somatic "O" and flagellar "H" antigens of S. dublin were measured in the serum of 43 pregnant heifers before intravenous or oral infection with S. dublin and in the serum of 21 uninfected control animals. The data from these animals were analysed statistically and a titre of 1/80, to both antigens, has been interpreted as of doubtful significance and a titre of 1/160 to both antigens, has been interpreted as significantly raised. Animals in which fetal infection occurred after challenge by either the intravenous or oral route developed significant increases in "H" and "O" titres indicating the value of measure "H" titres in the diagnosis of S. dublin abortion. In animals which were infected orally and in which infection appeared to be confined to the alimentary tract the "H" titre did not become significantly raised. Lack of correlation between antibody titres and faecal excretion of S. dublin and persistence of infection in carcasses confirms that the serum agglutination test is of no value in detecting latent carriers.