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The outspread of bacterial pathogens causing severe infections and spreading rapidly, especially among hospitalized patients, is worrying and represents a global public health issue. Current disinfection techniques are becoming insufficient to counteract the spread of these pathogens because they carry multiple antibiotic-resistance genes. For this reason, a constant need exists for new technological solutions that rely on physical methods rather than chemicals. Nanotechnology support provides novel and unexplored opportunities to boost groundbreaking, next-gen solutions. With the help of plasmonic-assisted nanomaterials, we present and discuss our findings in innovative bacterial disinfection techniques. Gold nanorods (AuNRs) immobilized on rigid substrates are utilized as efficient white light-to-heat transducers (thermoplasmonic effect) for photo-thermal (PT) disinfection. The resulting AuNRs array shows a high sensitivity change in refractive index and an extraordinary capability in converting white light to heat, producing a temperature change greater than 50 °C in a few minute interval illumination time. Results were validated using a theoretical approach based on a diffusive heat transfer model. Experiments performed with a strain of Escherichia coli as a model microorganism confirm the excellent capability of the AuNRs array to reduce the bacteria viability upon white light illumination. Conversely, the E. coli cells remain viable without white light illumination, which also confirms the lack of intrinsic toxicity of the AuNRs array. The PT transduction capability of the AuNRs array is utilized to produce white light heating of medical tools used during surgical treatments, generating a temperature increase that can be controlled and is suitable for disinfection. Our findings are pioneering a new opportunity for healthcare facilities since the reported methodology allows non-hazardous disinfection of medical devices by simply employing a conventional white light lamp.

The synthesis of carbamoyl phosphate by the mammalian multifunctional protein, CAD, involves the concerted action of the 40 kDa amidotransferase domain (GLN), that hydrolyzes glutamine and the 120 kDa synthetase (CPS) domain that uses the ammonia, thus produced, ATP and bicarbonate to make carbamoyl phosphate. The separately cloned GLN domain has very low activity due to a reduction in kcat and an increase in Km but forms a hybrid complex with the isolated Escherichia coli CPS subunit. The hybrid has full glutamine-dependent catalytic activity and a functional interdomain linkage. The mammalian-E. coli hybrid was used to investigate the functional consequence of replacing His336 and Glu338, two residues postulated to participate in catalysis as part of a catalytic triad. The mutant mammalian GLN domains formed stable complexes with the E. coli CPS subunit, but the catalytic activity was severely impaired. While the His336Asn mutant does not form measurable amounts of the gamma-glutamyl thioester, the steady state concentration of the intermediate with the Glu338Gly mutant was comparable to the wild type hybrid because both the rate of formation and breakdown of the thioester are reduced. This result is consistent with the postulated role of Glu338 in maintaining His336 in the optimal orientation for catalysis and suggests a mechanism for the GLN CPS functional linkage.

Carbamoyl-phosphate synthetase consists of an amidotransferase domain or subunit (GLN) that hydrolyzes glutamine and transfers the ammonia to the synthetase component (CPS) where the biosynthetic reaction occurs. The CPS domain is composed of two homologous subdomains, CPS.A and CPS.B, that catalyze different ATP-dependent reactions involved in carbamoyl phosphate synthesis. When the individual CPS.A and CPS.B subdomains were individually cloned and expressed in Escherichia coli (Guy, H. I., and Evans, D. R. (1996) J. Biol. Chem. 271, 13762-13769), they were found to be functionally equivalent and could each independently catalyze carbamoyl phosphate synthesis. The proposal was advanced that, although the monomers could catalyze the individual partial reactions, overall synthesis of carbamoyl phosphate required a homodimer of CPS.A or CPS.B. To test this hypothesis, the GLN-CPS.B dimer was reversibly dissociated at 1500 bar in a high pressure cell. Dissociation was accompanied by a loss of both glutamine- and ammonia-dependent CPSase activity. Activity was recovered once the protein was returned to atmospheric pressure. If the sample was cross-linked before exposure to high pressure, there was no dissociation and no loss of biosynthetic activity. In contrast, the bicarbonate-dependent ATPase and the carbamoyl phosphate-dependent ATP synthetase activities were largely unaffected by pressure-induced dissociation. These experiments confirmed the hypothesis that the synthesis of carbamoyl phosphate requires the concerted action of the two active sites within the homodimer.

Escherichia coli carbamyl-phosphate synthetase consists of two subunits that act in concert to synthesize carbamyl phosphate. The 40-kDa subunit is an amidotransferase (GLN subunit) that hydrolyzes glutamine and transfers ammonia to the 120-kDa synthetase subunit (CPS subunit). The enzyme can also catalyze ammonia-dependent carbamyl phosphate synthesis if provided with exogenous ammonia. In mammalian cells, homologous amidotransferase and synthetase domains are carried on a single polypeptide chain called CAD. Deletion of the 29-residue linker that bridges the GLN and CPS domains of CAD stimulates glutamine-dependent carbamyl phosphate synthesis and abolishes the ammonia-dependent reaction (Guy, H. I., and Evans, D. R. (1997) J. Biol. Chem. 272, 19906-19912), suggesting that the deletion mutant is trapped in a closed high activity conformation. Since the catalytic mechanisms of the mammalian and bacterial proteins are the same, we anticipated that similar changes in the function of the E. coli protein could be produced by direct fusion of the GLN and CPS subunits. A construct was made in which the intergenic region between the contiguous carA and carB genes was deleted and the sequences encoding the carbamyl-phosphate synthetase subunits were fused in frame. The resulting fusion protein was activated 10-fold relative to the native protein, was unresponsive to the allosteric activator ornithine, and could no longer use ammonia as a nitrogen donor. Moreover, the functional linkage that coordinates the rate of glutamine hydrolysis with the activation of bicarbonate was abolished, suggesting that the protein was locked in an activated conformation similar to that induced by the simultaneous binding of all substrates.

Saturation curves of activity versus concentration were determined for aspartate transcarbamylase from Escherichia coli (EC 2.1.3.2) for the substrate L-aspartate at saturating carbamyl phosphate (4.8 mM) in buffered solution at pH values from 6.0 to 12.0. Hill coefficients were obtained from the sigmoidal curves. At pH values from 7.8 to 9.1, where substrate inhibition causes difficulties in the Hill approximation, our kinetic scheme includes substrate inhibition and residual activity in the abortive enzyme-substrate complex. The plot of Hill coefficient versus pH has pKalpha values of 7.4 and 9.8 at the half-maximum positions of the curve which has a plateau from pH 8.1 to 9.1. These pKalpha values may be associated with functional groups involved in the allosteric transition which activates the enzyme. A plot of [S]0.5 versus pH shows a pKalpha of 8.5, which may belong to a residue either at or near the aspartate binding site. At 50 mM aspartate concentration the pH-rate profile shows maxima at pH values of 8.8 and 10.0 (cf. Weitzman, P.D.J., and Wilson, I.B.(1966)J. Biol. Chem. 2418 5481-5488, who used 100 mM aspartate). However, when the pH-dependent substrate inhibition is included, the calculated Vmax--H curve is bell-shaped like that of the isolated catalytic subunit.

Following our earlier determination of the three-dimensional structure of aspartate carbamoyltransferase (EC 2.1.3.2; carbamoylphosphate: L-aspartate carbamoyltransferase) to 5.5-A resolution [S. G. Warren, B. F. P. Edwards, D. R. Evans, D. C. Wiley & W. N. Lipscomb (1973) Proc. Nat. Acad. Sci. USA 70, 1117-1121], we report here, from a different crystal form, the three-dimensional structure at 5.9 A of this enzyme complexed with its allosteric inhibitor, cytidine triphosphate. Location of the major binding site of this inhibitor within each of the six regulatory chains is made secure by comparison of these results with those obtained upon binding of 5-iodocytidine triphosphate to the enzyme. Conformational changes in the aspartate carbamoyltransferase molecule when this inhibitor binds are described briefly at 5.9-A resolution.

A species produced by the reaction of aspartate transcarbamylase (C6R6) with 6 to 12 eq of p-hydroxymercuribenzoate was isolated by DEAE-Sephadex chromatography. Purified material was completely dissociated with mercurials and the relative amounts of catalytic (C) and regulatory (R) subunits were determined by three methods: (a) quantitative cellulose acetate electrophoresis; (b) Lowry analysis after separating the catalytic and regulatory subunits by sucrose gradient centrifugation; (c) dissociation of the species with sodium dodecyl sulfate and determination of the relative amounts of catalytic and regulatory chain by sodium dodecyl sulfate gel electrophoresis. All three methods gave consistent results, indicating that the molecule consists of 75% (by weight) catalytic chain and 25% regulatory chain. The molecular weight determined by gel filtration, sedimentation velocity, and sedimentation equilibrium experiments was found to be approximately 270,000. These observations establish that this species has the structure C6R4, and is produced by the release of a single regulatory dimer R2 from the intact aspartate transcarbamylase complex. This protein (C6R4) contains 20 cysteines and four zinc ions, consistent with the proposed subunit structure. The purified intermediate C6R4 contains no mercury. The parent molecule C6R6 can be reconstituted from C6R4 by incubation with isolated regulatory subunit (R2) in the presence of zinc and beta-mercaptoethanol. Titration of C6R4 yields an end point which corresponds to the addition of 1 mol of regulatory subunit (R2) per mol of C6R4. The intermediate is quite stable at neutral pH but tends to disproportionate into aspartate transcarbamylase and catalytic subunit after prolonged storage or at elevated pH. The kinetic properties of this species have been investigated. The specific activity of C6R4 is virtually identical with that of the native enzyme but the regulatory properties are substantially reduced. Both homotropic and heterotropic interactions are reduced but not abolished, indicating that the intact structure C6R6 is not required for the allosteric transitions involved in regulation.

A three-dimensional x-ray diffraction study of aspartate transcarbamylase to 5.5-angstrom resolution, with the aid of four isomorphous heavy atom derivatives, indicates the presence of a central aqueous cavity approximating an oblate spheroid about 25 by 50 by 50 angstroms in dimension, within a molecule about 90 by 110 by 110 angstroms in largest dimensions.

The allosteric enzyme, aspartate transcarbamoylase (EC 2.1.3.2), has previously been shown in our x-ray diffraction studies to have D(3)-32 symmetry. There are six catalytic (C) and six regulatory (R) chains in the molecular complex (R(6)C(6)). Our three-dimensional x-ray diffraction study of this enzyme (R32, a = 131 A, c = 200 A) at 5.5 A resolution shows a spatial arrangement of the two catalytic trimers C(3) above and below an equatorial belt of three regulatory dimers R(2). The molecule is about 110 x 110 x 90 A in largest dimensions, and is shown here to contain a large central aqueous cavity about 50 x 50 x 25 A in size. Location of the single sulfhydryl of each catalytic chain, and correlation of its reactivity with enzymatic activity in the molecule, suggests that the nearby active sites are most probably accessible from the central cavity, but probably not directly from the external solution. The most obvious access to the central cavity consists of six channels, each about 15 A in diameter, near the regulatory region. A component of the regulatory mechanism may be modulation of access of substrates through these channels.