Thoughts on thiolate tethering. Tribute and thanks to a teacher

A unique feature of P450 enzymes is in the presence of a thiolate ligand heme but its exact function in catalysis is a matter of debate. For P450 dependent monooxygenases the "active oxygen" complex seems to exist only as a transition state in which the thiolate ligand provides electron density in order to prevent pi-backbonding of the oxygen to the iron (-S-Fe-O(z.rad;)). The corresponding ground state (Compound I) would be a ferryl species (Fe(IV)z.dbnd6;O) with an electron hole either at the porphyrin or at the sulfur. Apart from this role we postulate that a second function is related to the electronic structure of Compound II as an electron acceptor and this property is shared among monooxygenases, thromboxane synthase, prostacyclin synthase, allene oxide synthase, P450(NOR(-)) and chloroperoxidase. As a common step in all P450 enzymes an extremely rapid electron uptake by Compound II allows that the primary substrate radicals are oxidized to cations which immediately combine with a neighbouring nucleophile. Thus "electron transfer" may substitute for "oxygen rebound" as the final step leading to product formation. The same principle also applies methane monooxygenases in which the role of the thiyl sulfur is replaced by a ferryl-oxyl entity.     

Synthesis of 3-tert-butylcatechol by an engineered monooxygenase

Recombinant Escherichia coli JM101 was used for the in vivo biocatalytic synthesis of 3-tert-butyl- catechol. The bacterial strain synthesized the laboratory-evolved variant HbpA(T2) of 2-hydroxybiphenyl 3-monooxygenase (HbpA, EC 1.14.13.44) from Pseudomonas azelaica HBP1. The mutant enzyme HbpA(T2) is able to hydroxylate 2-tert-butylphenol to the corresponding catechol, a reaction that is not catalyzed by the wild-type enzyme. The biotransformation was performed in a 3-L bioreactor for 24 h. To mitigate the toxicity of the 2-tert-butylphenol starting material, we applied a limited substrate feed. Continuous in situ product removal with the hydrophobic resin Amberlite XAD-4 was used to separate the product from culture broth. In addition, binding to the resin stabilized the product, which was important because 3-tert-butylcatechol is very labile in aqueous solution. The productivity of the process was 63 mg L(-1) h(-1) so that after 24 h, 3.0 g of 3-tert-butylcatechol were isolated. Down-stream processing consisted of two steps. First, bound 2-tert-butylphenol and 3-tert-butylcatechol were eluted from Amberlite XAD-4 with methanol. Second, the two compounds were separated over neutral aluminum oxide, which selectively binds the produced catechol but not the phenol substrate. The final purity of 3-tert-butylcatechol was greater than 98%.     

The use of microscale processing technologies for quantification of biocatalytic Baeyer-Villiger oxidation kinetics

Microscale processing techniques would be a useful tool for the rapid and efficient collection of biotransformation kinetic data as a basis for bioprocess design. Automated liquid handling systems can reduce labor intensity while the small scale reduces the demand for scarce materials such as substrate, product, and biocatalyst. Here we illustrate this concept by establishing the use of several microwell formats (96-round, 96-deep square and 24-round well microtiter plates) for quantification of the kinetics of the E. coli TOP10 [pQR239] resting cell catalyzed Baeyer-Villiger oxidation of bicyclo[3.2.0]hept-2en-6-one using glycerol as a source of reducing power. By increasing the biocatalyst concentration until the biotransformation rate was oxygen mass-transfer limited we can ensure that kinetic data collected are in the region away from oxygen limitation. Using a 96-round well plate the effect of substrate (bicyclo[3.2.0]hept-2en-6-one) concentration on the volumetric CHMO activity was examined and compared to data collected from 1.5-L stirred-tank experiments. The phenomenon and magnitude of substrate inhibition, observed at the larger scale, was accurately reproduced in the microwell format. We have used this as an illustrative example to demonstrate that under adequately defined conditions, automated microscale processing technologies can be used for the collection of quantitative kinetic data. Additionally, by using the experimentally determined stoichiometry for product formation and glycerol oxidation, we have estimated the maximum oxygen transfer rates as a function of well geometry and agitation rate. Oxygen-transfer rates with an upper limit of between 33 mmol. L(-1). h(-1) (based solely on product formation) and 390 mmol. L(-1). h(-1) (based on product formation and glycerol oxidation) were achieved using a 96-square well format plate shaken at 1300 rpm operated with a static surface area to volume ratio of 320 m(2). m(-3).     

Analysis of the roles of amino acid residues in the flavoprotein tryptophan 2-monooxygenase modified by 2-oxo-3-pentynoate: characterization of His338, Cys339, and Cys511 mutant enzymes

The flavoprotein tryptophan 2-monooxygenase catalyzes the oxidative decarboxylation of tryptophan to indoleacetamide. His338, Cys339, and Cys511 of the Pseudomonas savastanoi enzyme were previously identified as possible active-site residues by modification with 2-oxo-3-pentynoate ([G. Gadda, L.J. Dangott, W.H. Johnson Jr., C.P. Whitman, P.F. Fitzpatrick, Biochemistry 38 (1999) 5822-5828]). The H338N, C339A, and C511S enzymes have been characterized to determine the roles of these residues in catalysis. The steady-state kinetic parameters with both tryptophan and methionine decrease only slightly in the case of the H338N and C339A enzymes; the decrease in activity is greater for the C511S enzyme. Only in the case of the C511S enzyme do deuterium kinetic isotope effects on kinetic parameters indicate a significant change in catalytic rates. The structural bases for the effects of the mutations can be interpreted by identification of L-amino acid oxidase and tryptophan monooxygenase as homologous proteins.     

Geochemical Peculiarities of the Carbonate Constructions Formed during Microbial Oxidation of Methane under Anaerobic Conditions

The aragonite constructions of the Black Sea are formed in a stable anaerobic zone and are a perfect object to study the natural mechanism of anaerobic methane oxidation. The most probable pathway of methane oxidation is its methanogen-mediated reaction with bicarbonates, dissolved in seawater, with the formation of water and acetate, which is then consumed by other components of the anaerobic community. Comparison of the ¹³C values of carbonate minerals and organic matter once more demonstrated that the formation of the organic matter of biomass is accompanied by intense fractionation of carbon isotopes, as a result of which the total organic matter of biomass acquires an extremely light isotopic composition, characterized by ¹³C values as low as –83.8.     

Particulate methane monooxygenase from Methylosinus trichosporium is a copper-containing enzyme

Particulate methane monooxygenase (pMMO) has been exfoliated and isolated from membranes of the Methylosinus trichosporium IMV 3011. It appears that the stability of pMMO in the exfoliation process is increased with increasing copper concentration in the growth medium, but extensive intracytoplasmic membrane formed under higher copper concentration may inhibit the exfoliation of active pMMO from membrane. The highest total activity of purified pMMO is obtained with an initial concentration of 6 microM Cu in the growth medium. The purified MMO contains only copper and does not utilize NADH as electron donor. Treatment of purified pMMO with EDTA resulted in little change in copper level, suggesting that the copper in the pMMO is tightly bound with pMMO.     

Simulation on long-term operation of an anaerobic bioreactor for Korean food wastes

A mathematical model was formulated to simulate the long-term performance of an anaerobic bioreactor designed to digest Korean food wastes. The system variables of various decomposition steps were built into the model, which predicts the temporal characters of solid waste, and volatile fatty acid (VFA) in the reactor, and gas production in response to various input loadings and temperatures. The predicted values of VFA and gas production were found to be in good agreement with experimental observations in batch and repeated-input systems. Finally, long-term reactor performance was simulated with respect to the seasonal temperature changes from 5°C in winter to 25°C in summer at different food waste input loadings. The simulation results provided us with information concerning the success or failure of a process during long-term operation.     

Purification and characterization of two soluble acid invertase isozymes from Japanese pear fruit

Two isozymes (AIV I and AIV II) of soluble acid invertase (EC 3.2.1.26) were purified from Japanese pear fruit through procedures including (NH(4))(2)SO(4) precipitating, DEAE-Sephacel column chromatography, Concanavalin A (ConA)-Sepharose affinity chromatography, hydroxyapatite column chromatography and Mono Q HR 5/5 column chromatography. The specific activities of purified AIV I and AIV II were 2670 and 2340 (nkat/mg protein), respectively. AIV I was a monomeric enzyme of 80 kDa, while AIV II may be also a monomeric enzyme, which is easy to be cleaved to 52 kDa and 34 kDa polypeptide during preparation by SDS-PAGE. The Km values for sucrose of AIV I and AIV II were 3.33 and 4.58 mM, respectively, and optimum pH of both enzyme activities was pH 4.5.     

Fluxes of nitrous oxide and methane from drained peatlands following forest clear-felling in southern Finland

Drainage of waterlogged sites has been part of the normal forestry practice in Fennoscandia, the Baltic countries, the British Isles and in some parts of Russia since the early 20^th century, and currently, about 15 million hectares of peatlands and other wetlands have been drained for forestry purposes. The rate of forest clear-felling on drained peatlands will undergo a rapid increase in the near future, when a large number of these forests approach their regeneration age. A small-scale pilot survey was performed at two nutrient-rich and old peatland drainage areas in southern Finland to study if forest clear-felling has significant impacts on the exchange of nitrous oxide (N₂O) and methane (CH₄) between soil and atmosphere. The average N₂O emissions from the two drainage areas during three growing seasons following clear-felling were 945 and 246 g m^–2 d^–1. The corresponding CH₄ fluxes were –0.07 and –0.52 mg m^–2 d^–1. Clear-felling had impacts on the environmental factors known to affect the N₂O and CH₄ fluxes of peatlands, i.e. clear-felling raised the water table level and increased the peat temperature. However, no substantial changes in the fluxes of CH₄ following clear-felling were observed. The results concerning N₂O indicated a potential for increased emissions following clear-felling of drained peatland forests, but further studies are needed for a critical evaluation of the impacts of clear-felling on the fluxes of CH₄ and N₂O.     

Expression and characterization of human bifunctional peptidylglycine alpha-amidating monooxygenase

We report the purification and characterization of human bifunctional peptidylglycine alpha-amidating monooxygenase (the bifunctional PAM) expressed in Chinese hamster ovary cells. PAM is in charge of the formation of the C-terminal amides of biologically active peptides. The bifunctional PAM possesses two catalytic domains in a single polypeptide, peptidylglycine alpha-hydroxylating monooxygenase (PHM, EC 1.14.17.3) and peptidylamidoglycolate lyase (PAL, EC 4.3.2.5). By introducing a stop codon at 835 Glu, we were able to eliminate the membrane-spanning domain in the C-terminal region and succeeded in purifying a soluble form of bifunctional PAM that was secreted into the medium. Through a three-step purification procedure, we obtained 0.3mg of the purified PAM, which showed a single band at 91 kDa on SDS-PAGE, from 1L of monolayer culture medium. Metals contained in the purified PAM were analyzed and chemical modifications were performed to gain insight into the mechanism of the PAL reaction. Inductively coupled plasma detected 0.62 mol of Zn(2+) and 1.25 mol of Cu(2+) per mol of bifunctional PAM. Further, the addition of 1mM EDTA reduced the PAL activity by about 50%, but the decreased activity was recovered by the addition of an excess amount of Zn(2+). In a series of chemical modifications, phenylglyoxal almost completely eliminated the PAL activity and diethyl pyrocarbonate suppressed activity by more than 70%. These findings implied that Arg and His residues might play crucial roles during catalysis.