Quantitative analysis of residual protein contamination on reprocessed surgical instruments.

'Ready-for-use' instruments from surgical instrument trays were examined after routine cleaning and sterilization in a blinded study. These reprocessed instruments originated from five National Health Service hospital trust sterile service departments in England and Wales. Determination of residual protein and peptide contamination was carried out by acid stripping of the instrument surfaces, hydrolysis of the constituent amino acids and quantitative total amino acid analysis. One hundred and twenty instruments were analysed, and the median levels of residual protein contamination per instrument for the individual trays were 267, 260, 163, 456 and 756 microg. Scanning electron microscopy and energy dispersive X-ray spectroscopic analyses of the instruments showed that tissue deposits were localized on surfaces, but there was no significant correlation between overall protein soiling and instrument complexity. The highest levels of residual contamination were found on instruments used for tonsillectomy and adenoid surgery.

Elimination of transmissible spongiform encephalopathy infectivity and decontamination of surgical instruments by using radio-frequency gas-plasma treatment.

It has now been established that transmissible spongiform encephalopathy (TSE) infectivity, which is highly resistant to conventional methods of deactivation, can be transmitted iatrogenically by contaminated stainless steel. It is important that new methods are evaluated for effective removal of protein residues from surgical instruments. Here, radio-frequency (RF) gas-plasma treatment was investigated as a method of removing both the protein debris and TSE infectivity. Stainless-steel spheres contaminated with the 263K strain of scrapie and a variety of used surgical instruments, which had been cleaned by a hospital sterile-services department, were examined both before and after treatment by RF gas plasma, using scanning electron microscopy and energy-dispersive X-ray spectroscopic analysis. Transmission of scrapie from the contaminated spheres was examined in hamsters by the peripheral route of infection. RF gas-plasma treatment effectively removed residual organic residues on reprocessed surgical instruments and gross contamination both from orthopaedic blades and from the experimentally contaminated spheres. In vivo testing showed that RF gas-plasma treatment of scrapie-infected spheres eliminated transmission of infectivity. The infectivity of the TSE agent adsorbed on metal spheres could be removed effectively by gas-plasma cleaning with argon/oxygen mixtures. This treatment can effectively remove 'stubborn' residual contamination on surgical instruments.

Plasma cleaning of dental instruments.

The theoretical risk of prion transmission via surgical instruments is of current public and professional concern. These concerns are further heightened by reports of the strong surface affinity of the prion protein, and that the removal of organic material by conventional sterilization is often inadequate. Recent reports of contamination on sterilized endodontic files are of particular relevance given the close contact that these instruments may make with peripheral nerve tissue. In this paper, we report the effective use of a commercial gas plasma etcher in the cleaning of endodontic files. A representative sample of cleaned, sterilized, files was screened, using scanning electron microscopy and energy-dispersive X-ray analysis, to determine the level of contamination before plasma cleaning. The files were then exposed for a short-term to a low-pressure oxygen-argon plasma, before being re-examined. In all cases, the amount of organic material (in particular that which may have comprised protein) was reduced to a level below the detection limit of the instrument. This work suggests that plasma cleaning offers a safe and effective method for decontamination of dental instruments, thus reducing the risk of iatrogenic transmission of disease during dental procedures. Furthermore, whilst this study focuses on dental files, the findings indicate that the method may be readily extended to the decontamination of general surgical instruments.

Probing the NADPH-binding site of Escherichia coli flavodoxin oxidoreductase.

The structure of the Escherichia coli flavodoxin NADP(+) oxidoreductase (FLDR) places three arginines (R144, R174 and R184) in the proposed NADPH-binding site. Mutant enzymes produced by site-directed mutagenesis, in which each arginine was replaced by neutral alanine, were characterized. All mutants exhibited decreased NADPH-dependent cytochrome c reductase activity (R144A, 241.6 min(-1); R174A, 132.1 min(-1); R184A, 305.5 min(-1) versus wild type, 338.9 min(-1)) and increased K(m) for NADPH (R144A, 5.3 microM; R174A, 20.2 microM; R184A, 54.4 microM versus wild type, 3.9 microM). The k(cat) value for NADH-dependent cytochrome c reduction was increased for R174A (42.3 min(-1)) and R184A (50.4 min(-1)) compared with the wild type (33.0 min(-1)), consistent with roles for R174 and R184 in discriminating between NADPH/NADH by interaction with the adenosine ribose 2'-phosphate. Stopped-flow studies indicated that affinity (K(d)) for NADPH was markedly reduced in mutants R144A (635 microM) and R184A (2.3 mM) compared with the wild type (<5 microM). Mutant R184A displays the greatest change in pyridine nucleotide preference, with the NADH/NADPH K(d) ratio >175-fold lower than for wild-type FLDR. The rate constant for hydride transfer from NADPH to flavin was lowest for R174A (k(red)=8.82 s(-1) versus 22.63 s(-1) for the wild type), which also exhibited tertiary structure perturbation, as evidenced by alterations in CD and fluorescence spectra. Molecular modelling indicated that movement of the C-terminal tryptophan (W248) of FLDR is necessary to permit close approach of the nicotinamide ring of NADPH to the flavin. The positions of NADPH phosphates in the modelled structure are consistent with the kinetic data, with R174 and R184 located close to the adenosine ribose 2'-phosphate group, and R144 likely to interact with the nicotinamide ribose 5'-phosphate group.

Identification of the [Fe-S] cluster-binding residues of Escherichia coli biotin synthase.

The gene encoding Escherichia coli biotin synthase (bioB) has been expressed as a histidine fusion protein, and the protein was purified in a single step using immobilized metal affinity chromatography. The His(6)-tagged protein was fully functional in in vitro and in vivo biotin production assays. Analysis of all the published bioB sequences identified a number of conserved residues. Single point mutations, to either serine or threonine, were carried out on the four conserved (Cys-53, Cys-57, Cys-60, and Cys-188) and one non-conserved (Cys-288) cysteine residues, and the purified mutant proteins were tested both for ability to reconstitute the [2Fe-2S] clusters of the native (oxidized) dimer and enzymatic activity. The C188S mutant was insoluble. The wild-type and four of the mutant proteins were characterized by UV-visible spectroscopy, metal and sulfide analysis, and both in vitro and in vivo biotin production assays. The molecular masses of all proteins were verified using electrospray mass spectrometry. The results indicate that the His(6) tag and the C288T mutation have no effect on the activity of biotin synthase when compared with the wild-type protein. The C53S, C57S, and C60S mutant proteins, both as prepared and reconstituted, were unable to covert dethiobiotin to biotin in vitro and in vivo. We conclude that three of the conserved cysteine residues (Cys-53, Cys-57, and Cys-60), all of which lie in the highly conserved "cysteine box" motif, are crucial for [Fe-S] cluster binding, whereas Cys-188 plays a hitherto unknown structural role in biotin synthase.

Mechanism of 8-amino-7-oxononanoate synthase: spectroscopic, kinetic, and crystallographic studies.

8-Amino-7-oxononanoate synthase (also known as 7-keto-8-aminopelargonate synthase, EC 2.3.1.47) is a pyridoxal 5'-phosphate-dependent enzyme which catalyzes the decarboxylative condensation of L-alanine with pimeloyl-CoA in a stereospecific manner to form 8(S)-amino-7-oxononanoate. This is the first committed step in biotin biosynthesis. The mechanism of Escherichia coli AONS has been investigated by spectroscopic, kinetic, and crystallographic techniques. The X-ray structure of the holoenzyme has been refined at a resolution of 1.7 A (R = 18.6%, R(free) = 21. 2%) and shows that the plane of the imine bond of the internal aldimine deviates from the pyridine plane. The structure of the enzyme-product external aldimine complex has been refined at a resolution of 2.0 A (R = 21.2%, R(free) = 27.8%) and shows a rotation of the pyridine ring with respect to that in the internal aldimine, together with a significant conformational change of the C-terminal domain and subtle rearrangement of the active site hydrogen bonding. The first step in the reaction, L-alanine external aldimine formation, is rapid (k(1) = 2 x 10(4) M(-)(1) s(-)(1)). Formation of an external aldimine with D-alanine, which is not a substrate, is significantly slower (k(1) = 125 M(-)(1) s(-)(1)). Binding of D-alanine to AONS is enhanced approximately 2-fold in the presence of pimeloyl-CoA. Significant substrate quinonoid formation only occurs upon addition of pimeloyl-CoA to the preformed L-alanine external aldimine complex and is preceded by a distinct lag phase ( approximately 30 ms) which suggests that binding of the pimeloyl-CoA causes a conformational transition of the enzyme external aldimine complex. This transition, which is inferred by modeling to require a rotation around the Calpha-N bond of the external aldimine complex, promotes abstraction of the Calpha proton by Lys236. These results have been combined to form a detailed mechanistic pathway for AONS catalysis which may be applied to the other members of the alpha-oxoamine synthase subfamily.

Characterisation of flavodoxin NADP+ oxidoreductase and flavodoxin; key components of electron transfer in Escherichia coli.

The genes encoding the Escherichia coli flavodoxin NADP+ oxidoreductase (FLDR) and flavodoxin (FLD) have been overexpressed in E. coli as the major cell proteins (at least 13.5% and 11.4% of total soluble protein, respectively) and the gene products purified to homogeneity. The FLDR reduces potassium ferricyanide with a kcat of 1610.3 min(-1) and a Km of 23.6 microM, and cytochrome c with a kcat of 141.3 min(-1) and a Km of 17.6 microM. The cytochrome c reductase rate is increased sixfold by addition of FLD and an apparent Km of 6.84 microM was measured for the affinity of the two flavoproteins. The molecular masses of FLDR and FLD apoproteins were determined as 27648 Da and 19606 Da and the isoelectric points as 4.8 and 3.5, respectively. The mass of the FLDR is precisely that predicted from the atomic structure and indicates that residue 126 is arginine, not glutamine as predicted from the gene sequence. FLDR and FLD were covalently crosslinked using 1-ethyl-3(dimethylamino-propyl) carbodiimide to generate a catalytically active heterodimer. The midpoint reduction potentials of the oxidised/semiquinone and semiquinone/hydroquinone couples of both FLDR (-308 mV and -268 mV, respectively) and FLD (-254 mV and -433 mV, respectively) were measured using redox potentiometry. This confirms the electron-transfer route as NADPH-->FLDR-->FLD. Binding of 2' adenosine monophosphate increases the midpoint reduction potentials for both FLDR couples. These data highlight the strong stabilisation of the flavodoxin semiquinone (absorption coefficient calculated as 4933 M(-1) cm(-1) at 583 nm) with respect to the hydroquinone state and indicate that FLD must act as a single electron shuttle from the semiquinone form in its support of cellular functions, and to facilitate catalytic activity of microsomal cytochromes P-450 heterologously expressed in E. coli. Kinetic studies of electron transfer from FLDR/FLD to the fatty acid oxidase P-450 BM3 support this conclusion, indicating a ping-pong mechanism. This is the first report of the potentiometric analysis of the full E. coli NAD(P)H/FLDR/FLD electron-transfer chain; a complex critical to the function of a large number of E. coli redox systems.

The crystal structure of 8-amino-7-oxononanoate synthase: a bacterial PLP-dependent, acyl-CoA-condensing enzyme.

8-Amino-7-oxononanoate synthase (or 8-amino-7-ketopelargonate synthase; EC 2.3.1.47; AONS) catalyses the decarboxylative condensation of l-alanine and pimeloyl-CoA in the first committed step of biotin biosynthesis. We have cloned, over-expressed and purified AONS from Escherichia coli and determined the crystal structures of the apo and PLP-bound forms of the enzyme. The protein is a symmetrical homodimer with a tertiary structure and active site organisation similar to, but distinct from, those of other PLP-dependent enzymes whose three-dimensional structures are known. The critical PLP-binding lysine of AONS is located at the end of a deep cleft that allows access of the pantothenate arm of pimeloyl-CoA. A cluster of positively charged residues at the entrance to this cleft forms a putative diphosphate binding site for CoA. The structure of E. coli AONS enables identification of the key residues of the PLP-binding site and thus provides a framework with which to understand the biochemical mechanism, which is similar to that catalysed by 5-aminolevulinate synthase and two other alpha-oxoamine synthases. Although AONS has a low overall sequence similarity with the catalytic domains of other alpha-oxoamine synthases, the structure reveals the regions of significant identity to be functionally important. This suggests that the organisation of the conserved catalytic residues in the active site is similar for all enzymes of this sub-class of PLP-dependent enzymes and they share a common mechanism. Knowledge of the three-dimensional structure of AONS will enable characterisation of the structural features of this enzyme sub-family that are responsible for this important type of reaction.

Substrate binding and carboxylation by dethiobiotin synthetase--a kinetic and X-ray study.

BACKGROUND: The vitamin biotin is a ubiquitous prosthetic group of carboxylase and transcarboxylase enzymes. Biotin biosynthesis occurs by similar pathways in microorganisms and plants. The penultimate step in biotin biosynthesis, catalyzed by dethiobiotin synthetase (DTBS), involves a unique ATP-dependent N-carboxylation, resulting in formation of the ureido ring function of dethiobiotin. The first two steps of dethiobiotin formation, which is a complex, multistep enzymatic reaction, have been elucidated by a combination of X-ray crystallography and kinetic methods. RESULTS: The first step in catalysis by DTBS is the formation of an enzyme-substrate complex and the second is the enzymatic carboxylation of the bound substrate. Both steps are Mg2+ dependent. The kinetic constants in the presence and absence of Mg2+ have been measured and a set of X-ray structures determined at different stages of the reaction. The conformational changes in the active site of the enzyme, induced by Mg2+, substrate binding and substrate carboxylation, have been monitored crystallographically and are discussed. Sulfate ions bound to DTBS may mimic the behaviour of the alpha- and gamma-phosphates of ATP in Mg2+ binding and in the subsequent steps of the reaction. CONCLUSIONS: Mg2+ is an essential cation for both substrate binding and carbamate formation by DTBS, when sulfate is present. The conformational changes induced at the active site in the DTBS-substrate complex, when Mg2+ is present, are small yet highly significant and serve to optimize the interactions between substrate and enzyme. DTBS is active as a homodimer and the substrate-binding site straddles both monomers in the dimer. The carboxylation site is unambiguously identified as the N-7 amino group of the substrate, rather than the N-8 amino group, as previously suggested. The elongated nucleotide-binding loop (the P loop) binds both ATP and substrate in a manner which suggests that this feature may be of wider importance.

Mechanistic implications and family relationships from the structure of dethiobiotin synthetase.

BACKGROUND: Biotin is the vitamin essential for many biological carboxylation reactions, such as the conversion of acetyl-coenzyme A (CoA) to malonyl-CoA in fatty acid biosynthesis. Dethiobiotin synthetase (DTBS) facilitates the penultimate, ureido ring closure in biotin synthesis, which is a non-biotin-dependent carboxylation. DTBS displays no sequence similarity to any other protein in the database. Structural studies provide a molecular insight into the reaction mechanism of DTBS. RESULTS: We present the structure of DTBS refined to 1.80 A resolution with an R-factor of 17.2% for all terms plus unrefined data on the binding of the substrate, 7,8-diaminopelargonic acid and the product, dethiobiotin. These studies confirm that the protein forms a homodimer with each subunit folded as a single globular alpha/beta domain. The presence of sulphate ions in the crystals and comparisons with the related Ha-ras-p21 oncogene product are used to infer the ATP-binding site, corroborated by the difference electron density for the ATP analogue AMP-PNP. CONCLUSIONS: This study establishes that the enzyme active site is situated at the dimer interface, with the substrate binding to one monomer and ATP to the other. The overall fold of DTBS closely resembles that of three other enzymes, adenylosuccinate synthetase (purA), Ha-ras-p21, and nitrogenase iron protein, that are unrelated by sequence or function, indicating that DTBS is a member of a diverse family of enzymes.

Sequence and crystallization of Escherichia coli dethiobiotin synthetase, the penultimate enzyme of biotin biosynthesis.

The enzyme dethiobiotin synthetase (EC 6.3.3.3) has been cloned and over-expressed in Escherichia coli in such a way that milligram quantities are available. The purified enzyme has been subjected to a number of physical and chemical studies, sequenced and most notably it has been crystallized in a form that is suitable for X-ray structure determination. The cell dimensions are a = 72.8 A, b = 49.2 A, c = 61.4 A, beta = 106.2 degrees. The systematic absences are consistent with the monoclinic space group C2 with one polypeptide chain in the asymmetric unit.

Analysis of mutational lesions of acetate metabolism in Neurospora crassa by 13C nuclear magnetic resonance.

The adaptation of Neurospora crassa mycelium to growth on acetate as the sole carbon source was examined by using 13C nuclear magnetic resonance. Extracts were examined by nuclear magnetic resonance at various times after transfer of the mycelium from medium containing sucrose to medium containing [2-13C]acetate as the sole carbon source. The label was initially seen to enter the alanine, glutamate, and glutamine pools, and after 6 h 13C-enriched trehalose was evident, indicating that gluconeogenesis was occurring. Analysis of the isotopomer ratios in the alanine and glutamate-glutamine pools indicated that substantial glyoxylate cycle activity became evident between 2 and 4 h after transfer. Immediately after transfer of the mycelium to acetate medium, the alanine pool increased to about four times its previous level, only a small fraction of which was enriched with 13C. The quantity of 13C-enriched alanine remained almost constant between 2 and 7.5 h after the transfer, whereas the overall alanine pool decreased to its original level. The selective catabolism of the unenriched alanine leads us to suggest that the alanine pool is partitioned into two compartments during adaptation. Two acetate-nonutilizing mutants were also studied by this technique. An acu-3 strain, deficient for isocitrate lyase (EC 4.1.3.1) activity, showed metabolic changes consistent with this lesion. An acp strain, previously thought to be deficient in an inducible acetate permease, took up [2-13C]acetate but showed no evidence of glyoxylate cycle activity despite synthesizing the necessary enzymes; the lesion was therefore reinterpreted.

Biosynthesis of porphyrins and corrins. 1. 1H and 13C NMR spectra of (hydroxymethyl)bilane and uroporphyrinogens I and III.

High-field NMR spectroscopic methods have been applied to study the reactions catalyzed by porphobilinogen (PBG) deaminase and uroporphyrinogen III (uro'gen III) cosynthase, which are the enzymes responsible for the formation of the porphyrin macrocycle. The action of these enzymes in the conversion of PBG, [2,11-13C]PBG, and [3,5-13C]PBG to uro'gens I and III has been followed by 1H and 13C NMR, and assignments are presented. The principal intermediate that accumulated was the correspondingly labeled (hydroxymethyl)bilane (HMB), the assignments for which are also presented.

Microbiological applications of NMR spectroscopy. Part 1.

In the first part of this two-part review, the basic concepts of high resolution nuclear magnetic resonance (NMR) methods are described. In addition, the applications of 31P-NMR in the study of microbial metabolism are outlined.

13C NMR studies of acetate metabolism during sporulation of Saccharomyces cerevisiae.

The sporulation of Saccharomyces cerevisiae in the presence of [2-13C]acetate was studied by 13C NMR spectroscopy. The fate of 13C label was analyzed in vivo and in cell extracts. During the first 4 hr of sporulation the major metabolite produced from [2-13C]acetate utilization was glutamate. From the labeling pattern observed it is concluded that both the tricarboxylic acid cycle and the glyoxylate cycle are operating. After about 4 hr trehalose is made. Comparison of the doublet/singlet ratios for C-1,1(1) and C-6,6(1) of trehalose shows a steady drop in the ratio of C-1, C-2-coupled species over trehalose labeled only at C-1 in the C-1, 2 segment of the molecule. The negative correlation of this ratio with that for the C-5, 6 segment indicates a cycling of glucose through the hexose monophosphate shunt. Subsequently fatty acid biosynthesis commences. Large amounts of saturated fatty acid were made. There were conspicuous differences observed in the metabolism of [2-13C]acetate between sporulating and vegetatively growing cells.

Applications of 13C NMR to metabolic studies.

It is our view that the above examples, most of which have been published in the last two years, represent the beginnings of a new field and that in spite of major problems in instrumentation and programming, the essential feasibility of using enriched 13C substrates for metabolic processes as a noninvasive technique has been firmly established. With the caveat that a rigorous protocol for signal assignment must be devised, we can look forward to many applications of this method in a wide range of biological systems.