The methanol-oxidizing system of Methylobacterium extorquens AM1 reconstituted with purified constituents

The electron transport system (with cytochrome aa3) coupled to the oxidation of methanol in Methylobacterium extorquens AM1 (former Pseudomonas AM1) was reconstituted with highly purified constituents of the system. A mixture of 2.7 microM methanol dehydrogenase, 3.2 microM cytochrome cH, and 71 nM cytochrome c oxidase (= cytochrome aa3) consumed oxygen at a lower rate in the presence of methanol, while its activity was enhanced 3-fold by the addition of 1.4 microM cytochrome cL (74 mol of O2 consumed/mol of heme a of cytochrome c oxidase per min). Further addition of amicyanin to the above mixture did not affect the activity. Although ammonium ion greatly activated the activity of methanol dehydrogenase, the ion had little effect on the oxygen consumption activity of the above mixture. On the basis of the results obtained in the present study, an electron transport system is proposed for the oxidation of methanol in M. extorquens AM1.     

Cell-free translation reconstituted with purified components

We have developed a protein-synthesizing system reconstituted from recombinant tagged protein factors purified to homogeneity. The system was able to produce protein at a rate of about 160 microg/ml/h in a batch mode without the need for any supplementary apparatus. The protein products were easily purified within 1 h using affinity chromatography to remove the tagged protein factors. Moreover, omission of a release factor allowed efficient incorporation of an unnatural amino acid using suppressor transfer RNA (tRNA).     

Uptake of methylamine and methanol by Pseudomonas sp. strain AM1.

The uptake of methylamine and of methanol by the facultative methylotroph Pseudomonas sp. strain AM1 was investigated. It was found that this organism possesses two uptake systems for methylamine. One of these operates when methylamine is the sole source of carbon, nitrogen, and energy. It has a Km of 1.33 X 10(-4) M and a Vmax of 67 nmol/min per mg of cells (dry weight). The other system, found when methylamine is the sole nitrogen source only, has a Km of 1.2 X 10(-5) M and a Vmax of 8.9 nmol/min per mg of cells (dry weight). Both uptake systems were severely inhibited by azide, cyanide, carbonyl cyanide-m-chlorophenyl hydrazone, and N-ethylmaleimide, but only the high-affinity system was inhibited by ammonium ions with a Ki of 7.7 mM. Both systems were susceptible to osmotic shock treatment, competitively inhibited by ethylamine, and unaffected by most amino acids. Methanol uptake showed a Km of 4.8 microM and a Vmax of 60.6 nmol/min per mg of cells (dry weight) and was not inhibited by osmotic shock treatment. Azide, cyanide, and N-ethylmaleimide curtailed uptake, but carbonyl cyanide-m-chlorophenyl hydrazone merely reduced the rate of uptake. A methanol dehydrogenase mutant, M15A, was unable to take up methanol. It is proposed that methanol diffuses into the cell where it is rapidly oxidized by methanol dehydrogenase.     

The prosthetic group of methylamine dehydrogenase from Pseudomonas AM1: evidence for a quinone structure

The g-value and linewidth of ESR spectra of methylamine dehydrogenase (primary-amine:(acceptor) oxidoreductase (deaminating) EC 1.4.99.-) and methanol dehydrogenase (alcohol:(acceptor) oxidoreductase, EC 1.1.99.8) are very similar. This similarity is also reflected in electron-nuclear double resonance (ENDOR) results, the coupling constants of two protons in one enzyme equalling those in the other. The presence of a third proton in the ENDOR spectrum of methylamine dehydrogenase suggests a different structure or a different kind of interaction which can be related to the finding that the resolved ROSTHETIC GROUP IS PROTEIN-BOUND. The bound prosthetic group has a high redox-potential, supporting the conclusion from the ESR and ENDOR results that it is a quinone derivative.     

Purification and properties of an amine dehydrogenase from Pseudomonas AM1 and its role in growth on methylamine

1. Whole cells of Pseudomonas AM1 grown on methylamine oxidize methylamine, formaldehyde and formate. Crude extracts oxidize methylamine only if supplemented with phenazine methosulphate. 2. By using a spectrophotometric assay, the methylamine-oxidizing enzyme has been purified 20-fold in 31% yield. 3. The enzyme is a dehydrogenase, unable to utilize oxygen, NAD, NADP, flavines or menadione as electron acceptors, but able to utilize phenazine methosulphate, ferricyanide, cytochrome c or brilliant cresyl blue. 4. The enzyme is non-specific, readily oxidizing aliphatic monoamines and diamines, histamine and ethanol-amine. Secondary and tertiary amines, quaternary ammonium salts and aromatic amines are not oxidized. 5. The pH optima for methylamine, n-pentylamine and putrescine are respectively 7.6, 8.0 and 8.5. 6. The K(m) value for methylamine is 5.2mum and that for phenazine methosulphate 56mum. 7. The enzyme will withstand heating for 15min. at 80 degrees without loss of activity, but is inactivated at higher temperatures. It is not inactivated by any pH value between 2.6 and 10.6. 8. The dehydrogenase is inhibited by semicarbazide (K(i) 3.35mum), isoniazid (K(i) 1.17mum), cuprizone (K(i) 0.49mum), p-chloromercuribenzoate (K(i) 0.45mm) and quinacrine (K(i) 12.1mm). 9. The enzyme is absent from succinate-grown cells, and, during adaptation from succinate to methylamine, activity appears before growth on methylamine begins.     

Archaeal DNA uracil repair via direct strand incision: A minimal system reconstituted from purified components

Hydrolytic deamination of DNA cytosine residues results in U/G mispairs, pre-mutagenic lesions threatening long-term genetic stability. Hence, DNA uracil repair is ubiquitous throughout all extant life forms and base excision repair, triggered by a uracil DNA glycosylase (UDG), is the mechanistic paradigm adopted, as it seems, by all bacteria and eukaryotes and a large fraction of archaea. However, members of the UDG superfamily of enzymes are absent from the extremely thermophilic archaeon Methanothermobacter thermautotrophicus ΔH. This organism, as a hitherto unique case, initiates repair by direct strand incision next to the DNA-U residue, a reaction catalyzed by the DNA uridine endonuclease Mth212, an ExoIII homologue. To elucidate the detailed mechanism, in particular to identify the molecular partners contributing to this repair process, we reconstituted DNA uracil repair in vitro from only four purified enzymes of M. thermautotrophicus ΔH. After incision at the 5′-side of a 2′-d-uridine residue by Mth212 DNA polymerase B (mthPolB) is able to take over the 3′-OH terminus and carry out repair synthesis generating a 5′-flap structure that is resolved by mthFEN, a 5′-flap endonuclease. Finally, DNA ligase seals the resulting nick. This defines mechanism and minimal enzymatic requirements of DNA-U repair in this organism.     

Genetic organization of methylamine utilization genes from Methylobacterium extorquens AM1.

An isolated 5.2-kb fragment of Methylobacterium extorquens AM1 DNA was found to contain a gene cluster involved in methylamine utilization. Analysis of polypeptides synthesized in an Escherichia coli T7 expression system showed that five genes were present. Two of the genes encoded the large and small subunits of methylamine dehydrogenase, and a third encoded amicyanin, the presumed electron acceptor for methylamine dehydrogenase, but the function of the other two genes is not known. The order on the 5.2-kb fragment was found to be large-subunit gene, the two genes of unknown function, small-subunit gene, amicyanin gene. The gene for azurin, another possible electron acceptor in methylamine oxidation, does not appear to be present within this cluster of methylamine utilization genes.     

The small-subunit polypeptide of methylamine dehydrogenase from Methylobacterium extorquens AM1 has an unusual leader sequence.

The nucleotide sequence for the N-terminal region of the small subunit of methylamine dehydrogenase from Methylobacterium extorquens AM1 has revealed a leader sequence that is unusual in both its length and composition. Gene fusions to lacZ and phoA show that this leader sequence does not function in Escherichia coli but does function in M. extorquens AM1.     

The two cytochromes c in the facultative methylotroph Pseudomonas AM1

It was previously suggested that there is only one soluble cytochrome c in Pseudomonas AM1, having a molecular weight of 20000, a redox midpoint potential of about +260mV and a low isoelectric pint [Anthony (1975) Biochem. J. 146, 289–298; Widdowson & Anthony (1975) Biochem. J. 152, 349–356]. A more thorough examination of the soluble fraction of methanol-grown Pseudomonas AM1 has now revealed the presence of two different cytochromes c. These were both purified to homogeneity by acid treatment, ion-exchange chromatography, gel filtration, chromatography on hydroxyapatite and preparative isoelectric focusing. Molecular weights were determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis; midpoint redox potentials were determined directly by using platinum and calomel electrodes; isoelectric points were estimated by electrophoresis and by the behaviour of the two cytochromes on ion-exchange celluloses. The more abundant cytochrome c_H (λ_max. 550.5nm) had a low molecular weight (11000), a midpoint potential of about +294mV and a high isoelectric point, not being adsorbed on DEAE-cellulose in 20mm-Tris/HCl buffer, pH8.0. The less abundant cytochrome c_L (λ_max. 549nm) was about 30% of the total; it had a high molecular weight (20900), a midpoint potential of about +256mV and a low isoelectric point, binding strongly to DEAE-cellulose in 20mm-Tris/HCl buffer, pH8.0. The pH-dependence of the midpoint redox potentials of the two cytochromes c were very similar. There were four ionizations affecting the redox potentials in the pH range studied (pH4.0–9.5), two in the oxidized form (pK values about 3.5 and 5.5) and two in the reduced form (pK values about 4.5 and 6.5), suggesting that the ionizing groups involved may be the two propionate side chains of the haem. Neither of the cytochromes c was present in mutant PCT76, which was unable to oxidize or grow on C₁ compounds, although still able to grow well on multicarbon compounds such as succinate. Whether or not these two cytochromes c have separate physiological functions is not yet certain.     

The stabilisation of purified, reconstituted P-glycoprotein by freeze drying with disaccharides

The drug efflux pump P-glycoprotein (P-gp) (ABCB1) confers multidrug resistance, a major cause of failure in the chemotherapy of tumours, exacerbated by a shortage of potent and selective inhibitors. A high throughput assay using purified P-gp to screen and characterise potential inhibitors would greatly accelerate their development. However, long-term stability of purified reconstituted ABCB1 can only be reliably achieved with storage at −80 °C. For example, at 20 °C, the activity of ABCB1 was abrogated with a half-life of <1 day. The aim of this investigation was to stabilise purified, reconstituted ABCB1 to enable storage at higher temperatures and thereby enable design of a high throughput assay system. The ABCB1 purification procedure was optimised to allow successful freeze drying by substitution of glycerol with the disaccharides trehalose or maltose. Addition of disaccharides resulted in ATPase activity being retained immediately following lyophilisation with no significant difference between the two disaccharides. However, during storage trehalose preserved ATPase activity for several months regardless of the temperature (e.g. 60% retention at 150 days), whereas ATPase activity in maltose purified P-gp was affected by both storage time and temperature. The data provide an effective mechanism for the production of resilient purified, reconstituted ABCB1.     

The microbial metabolism of C1 compounds. The electron-transport chain of Pseudomonas AM1

Pseudomonas AM1, Hyphomicrobium X and Pseudomonas MS all contain cytochrome a/a(3) and a b-type cytochrome able to react with CO. Pseudomonas AM1 and Hyphomicrobium X also have a CO-binding cytochrome c. The purified cytochrome c (redox potential 0.26V) of Pseudomonas AM1 was not susceptible to oxidation by molecular oxygen. CO reacted slowly with the reduced form giving a CO difference spectrum with a peak at 412nm and troughs at 420nm and 550nm. Similar results were obtained with the cytochrome c of Hyphomicrobium (aerobically grown or anaerobically grown with nitrate) and with that of Pseudomonas extorquens. The results given in the present paper are incompatible with an oxygenase or oxidase function for the soluble cytochrome c of methylotrophs. Studies with whole cells of Pseudomonas AM1 and a cytochrome c-deficient mutant have demonstrated that cytochrome b (redox potential 0.009V) is the first cytochrome in the electron-transport chain for oxidation of all substrates except methanol (and ethanol) whose oxidation does not involve this cytochrome. All substrates are usually oxidized by way of cytochrome c and cytochrome oxidase (cytochrome a/a(3)), but there is an alternative route for the reduction of cytochrome a/a(3) in the mutant lacking cytochrome c. Results of experiments on cyanide inhibition of respiration and cytochrome oxidation support the suggestion that the susceptibility of cytochrome b to oxidation by molecular oxygen (reflected in its ability to react with CO) is probably irrelevant to the normal physiology of Pseudomonas AM1.     

Efficient protein selection based on ribosome display system with purified components

Using the PURE (Protein synthesis Using Recombinant Elements) system, we developed an efficient and highly controllable ribosome display method for selection of functional protein. The PURE system is composed of purified factors and enzymes that are responsible for gene expression in Escherichia coli. We performed the detailed analyses and optimization of the ribosome display system and demonstrated the formation of stable mRNA/ribosome/polypeptide ternary complexes. As complex formation is fundamental to successful ribosome display, these improvements resulted in a dramatic increase in the mRNA recovery rate. As a result, a approximately 12,000-fold enrichment of single-chain antibody (scFv) cDNA was achieved in a single round of selection. Specific selection of scFv mRNA from a 1:10(10) dilution in competitor mRNA was achieved with only three rounds of affinity selection. These findings, together with the results in the accompanying paper [T. Matsuura, H. Yanagida, J. Ushioda, I. Urabe, T. Yomo, Nascent chain, RNA, and ribosome complexes generated by pure translation system (see the accompanying paper).], demonstrate that the PURE system can provide a basis for reliable and reproducible ribosome display.     

Computer controlled fed-batch fermentation of the methylotroph Pseudomonas AM1

Summary A simple on-line computer control strategy based on dissolved oxygen level readings has been developed to control methanol addition during a fermentation of the methylotroph Pseudomonas AM1. This strategy has led to significant and reproducible improvements in the performance of the fermentation.     

The autoreducible cytochromes c of the methylotrophs Methylophilus methylotrophus and Pseudomonas AM1.

The two types of soluble cytochrome c (cytochrome cH and cytochrome cL) found in methylotrophs are completely distinct proteins; one type is not a dimer or degradation product of the other. Free thiol groups are probably not involved in the unusually rapid autoreduction of the cytochromes at high pH. The axial ligands to the haem iron, histidine and methionine, are the same as in other low-spin cytochromes c. The methionine ligand is displaced at high pH by an alternative strong-field ligand. This displacement does not occur on reduction of cytochrome cL by methanol dehydrogenase, but this does not rule out the possibility that the autoreduction mechanism is involved in the interaction of the dehydrogenase and cytochrome c.     

Association of purified thyroid lysosomes to reconstituted microtubules

We report the characteristics of the interaction between reconstituted microtubules and purified thyroid lysosomes. Microtubules were extracted from pig brain by temperature-dependent assembly-disassembly and labelled with 125I by conjugation with the Bolton-Hunter reagent. Thyroid lysosomes were purified from pig thyroid by isopycnic centrifugation on Percoll gradients. The formation of microtubule-lysosome complexes has been studied by electron microscopy, using negative staining, and by differential centrifugation. The association of lysosomes to microtubules is time- and temperature-dependent (between 25 degrees C and 37 degrees C). The rate of microtubule-lysosome complex formation is related to the concentration of lysosomes. The higher the lysosome concentration is, the higher also is the rate of the interaction. Changes in microtubule concentration merely alter the amount of complex formed; there is a linear relationship between the amount of complexes and the microtubule concentration. However, lysosomes seem to possess a limited number of 'microtubule-binding sites', since a saturation of the complex formation can be obtained at high microtubule concentration. Two main types of complex have been observed by electron microscopy on negatively stained samples; simple complexes composed of a lysosome in close contact with a microtubule and complexes formed by a lysosome surrounded by several microtubules. The formation of microtubule-lysosome complexes was totally inhibited in the presence of 100 microM N-ethylmaleimide; the rate of the interaction was slightly increased in the presence of dithiothreitol (25-100 microM). The interaction we describe here in an acellular system might be relevant to the association of lysosomes to microtubules observed in intact cells (Collot, M., Louvard D. and Singer S.J. (1984) Proc. Natl. Acad. Sci. USA 81, 788-792) and will constitute a useful model to study the regulation mechanisms of microtubule-vesicle interaction.