The crystal structure of phenol hydroxylase in complex with FAD and phenol provides evidence for a concerted conformational change in the enzyme and its cofactor during catalysis.

BACKGROUND: The synthesis of phenolic compounds as by-products of industrial reactions poses a serious threat to the environment. Understanding the enzymatic reactions involved in the degradation and detoxification of these compounds is therefore of much interest. Soil-living yeasts use flavin adenine dinucleotide (FAD)-containing enzymes to hydroxylate phenols. This reaction initiates a metabolic sequence permitting utilisation of the aromatic compound as a source of carbon and energy. The phenol hydroxylase from Trichosporon cutaneum hydroxylates phenol to catechol. Phenol is the best substrate, but the enzyme also accepts simple hydroxyl-, amino-, halogen- or methyl-substituted phenols. RESULTS: The crystal structure of phenol hydroxylase in complex with FAD and phenol has been determined at 2.4 A resolution. The structure was solved by the MIRAS method. The protein model consists of two homodimers. The subunit consists of three domains, the first of which contains a beta sheet that binds FAD with a typical beta alpha beta nucleotide-binding motif and also a fingerprint motif for NADPH binding. The active site is located at the interface between the first and second domains; the second domain also binds the phenolic substrate. The third domain contains a thioredoxin-like fold and is involved in dimer contacts. The subunits within the dimer show substantial differences in structure and in FAD conformation. This conformational flexibility allows the substrate to gain access to the active site and excludes solvent during the hydroxylation reaction. CONCLUSIONS: Two of the domains of phenol hydroxylase are similar in structure to p-hydroxybenzoate hydroxylase. Thus, phenol hydroxylase is a member of a family of flavin-containing aromatic hydroxylases that share the same overall fold, in spite of large differences in amino acid sequences and chain length. The structure of phenol hydroxylase is consistent with a hydroxyl transfer mechanism via a peroxo-FAD intermediate. We propose that a movement of FAD takes place in concert with a large conformational change of residues 170-210 during catalysis.

Sources and nature of heterogeneity in recombinant phenol hydroxylase derived from the basidiomycetous soil yeast Trichosporon cutaneum.

Preparations of the dimeric flavoenzyme phenol hydroxylase derived from Trichosporon cutaneum were found to contain an active tetrameric form when the enzyme was produced in Escherichia coli. The relative content of the tetramer was estimated from scans of silver-stained native PAGE gels and/or size-exclusion chromatography (SEC). Proportions of up to 22% of the enzyme protein, depending on the growth temperature and the level of added inducer, were observed in independent cultures as well as in purified preparations. No tetramer was ever seen in cell extracts or purified preparations from T. cutaneum. Traces of higher multimers and of possibly deamidated species were also detected in preparations of the recombinant enzyme. The rate of enzyme production seems to be the major factor in promoting formation of the tetramer, whereas the specific growth rate of the fermentor culture appears to be of minor importance. The dimeric and the tetrameric forms were purified using either SEC or ion-exchange chromatography as a final step. The two purified species did not interchange under a variety of conditions, indicating that they are not undergoing rapid equilibria. The FAD of either form, as isolated by SEC, was present to a lower-than-expected extent of 2 equiv/dimer. However, by removing FAD and reconstituting the resulting apoproteins with the cofactor, the FAD content could be increased to 2 equiv. in the dimer and 3 equiv. in the tetramer. Both reconstituted forms exhibited absorption spectra identical with that of phenol hydroxylase from T. cutaneum as well as that of the recombinant enzyme. All spectra were equally perturbed by one equivalent of phenol per enzyme-attached FAD. The ratio of specific activities of the dimeric and the tetrameric forms was, however, lower than expected from the ratio of their FAD contents. The results are compatible with the notion that the tetramer consists of a native phenol hydroxylase dimer associated with a non-native one with a decreased ability to bind FAD, either in one or both of its constituent 'monomers'.

Derivatives of 3-digitoxigenone amidinohydrazone: synthesis and effect on the digitalis receptor of several species. Part 7: Compounds with positive inotropic activity.

2-Digitoxigenone amidinohydrazone (1), a compound with known digitalis-like activity, and Schiff bases 2-11 of 3-digitoxigenone were synthesized and tested pharmacologically in order to further determine possible structural requirements at the 3-position of digitalis compounds. The inotropic activity was screened using guinea-pig atria, and the interaction with the digitalis receptor was further examined using [3H]ouabain binding to cardiac membranes from guinea pig, rat, pit and man. All compounds revealed activities intermediate between 3-digitoxigenone and ouabain, and the potency of the derivatives covered approximately one order of magnitude. The absolute potency varied among species, but the rank order of potency was rather similar, yielding good correlations between species. This indicates no pronounced preference of these compounds for a particular (Na+/K+)-ATPase isoform of any of the species studied.

A fermentor culture for production of recombinant phenol hydroxylase.

Fermentor cultures using the fed-batch technique produced the FAD-containing enzyme phenol hydroxylase (EC 1.14.13.7) originated in the lower eukaryote Trichosporon cutaneum, but expressed in Escherichia coli under the control of the tac promoter. At 30 degrees C and isopropyl beta-D-thiogalactopyranoside (IPTG) concentrations of 0.5-2 mM, the enzyme protein was expressed to high cellular content, but aggregated into inclusion bodies. At 25 degrees C similar levels of enzyme protein were synthesized after induction with 0.05 mM IPTG, but a soluble, active enzyme was obtained. The active enzyme was produced at up to 45% of total protein and constituted more than 50% of soluble protein. The total yield was 5 g x liter-1. The FAD content of the cells increased after induction at a rate not limiting the formation of active enzyme. The enzyme was purified in two chromatographic steps. The N-terminal amino acid residue and the kinetic properties of the purified recombinant enzyme were similar to those reported for the enzyme from T. cutaneum.

Crystallization and preliminary X-ray analysis of phenol hydroxylase from Trichosporon cutaneum.

Recombinant phenol hydroxylase from the soil yeast Trichosporon cutaneum has been crystallized with PEG 4000 as precipitant. The crystals are monoclinic, space group P2(1) with cell dimensions a = 101.8 A, b = 153.0 A, c = 116.0 A and beta = 114.8 degrees. The crystal asymmetric unit most likely contains two dimers of phenol hydroxylase corresponding to a packing density in the crystals of 2.54 A 3/Da. The self-rotation function is consistent with the packing of two dimers in the asymmetric unit. The observed diffraction pattern extends beyond 2.8 A resolution and the crystals are well suited for structural analysis by X-ray diffraction methods.

Phenol hydroxylase from Trichosporon cutaneum: gene cloning, sequence analysis, and functional expression in Escherichia coli.

A cDNA clone encoding phenol hydroxylase from the soil yeast Trichosporon cutaneum was isolated and characterized. The clone was identified by hybridization screening of a bacteriophage lambda ZAP-based cDNA library with an oligonucleotide probe which corresponded to the N-terminal amino acid sequence of the purified enzyme. The cDNA encodes a protein consisting of 664 amino acids. Amino acid sequences of a number of peptides obtained by Edman degradation of various cleavage products of the purified enzyme were identified in the cDNA-derived sequence. The phenol hydroxylase cDNA was expressed in Escherichia coli to yield high levels of active enzyme. The E. coli-derived phenol hydroxylase is very similar to the T. cutaneum enzyme with respect to the range of substrates acted upon, inhibition by excess phenol, and the order of magnitude of kinetic parameters in the overall reaction. Southern blot analysis revealed the presence of phenol hydroxylase gene-related sequences in a number of T. cutaneum and Trichosporon beigelii strains and in Cryptococcus elinovii but not in Trichosporon pullulans, Trichosporon penicillatum, or Candida tropicalis.

The N-terminal amino acid sequence of phenol hydroxylase contains a dinucleotide-binding sequence motif.

The N-terminal sequence of phenol hydroxylase from Trichosporon cutaneum was determined by Edman degradation of the integral protein and of fragments obtained by hydroxylamine cleavage and by digestion with Staphylococcus V8 protease. A continuous sequence of 80 residues from the N terminus was determined: TKYSESYCDV10, LIVGAGPAGL20 MAARVLSEYV30 RQKPDLKVRI40 IDKRSTKVYN50 GQADGLQCRT60 LESLKNLRLA70 DKIXSEXNDM80. A single N-terminal sequence was detected, suggesting two identical subunits in the dimeric enzyme. We suggest the occurrence of an FAD-binding site near the N terminus. The C-terminal sequence is -LSTA, as determined by carboxypeptidase digestion.

Arginyl residues in the NADPH-binding sites of phenol hydroxylase.

Phenol hydroxylase was inactivated by the arginine reagents 2,3-butanedione, 1,2-cyclohexanedione, and phenylglyoxal. The cosubstrate NADPH, as well as NADPH+ and several analogues thereof, protected the enzyme against inactivation. Phenol did not protect the activity against any of the reagents used, nor did modification by 2,3-butanedione affect the binding of phenol. We propose the presence of arginyl residues in the binding sites for the adenosine phosphate part of NADPH.

Amino acid sequences around the pyridoxal-5'-phosphate-binding sites of phenol hydroxylase.

Phenol hydroxylase was labelled with pyridoxal 5'-phosphate. A radioactive label was introduced by using sodium boro[3H]hydride to reduce the initially formed Schiff's base. The labelled enzyme was digested with Staphylococcus V8 protease. Labelled peptides were isolated and their sequences were determined. The label could be located to three different lysyl residues. Sequence similarities with the known structures of p-hydroxybenzoate hydroxylase and glutathione reductase are discussed. The positions of the labelled sequences, relative to the bound ligands at the active site, are proposed on the basis of such sequence similarities.

Activation enthalpies and pH dependence of phenol hydroxylase from Trichosporon cutaneum, in vitro and in situ.

The effect of pH and temperature on phenol hydroxylase in vitro was compared to the corresponding effect on the enzyme in situ, in permeabilized cells. Activation enthalpies in situ were about 75-80% of those in vitro, in both cases decreasing with increasing pH (6.0-8.5). The order of addition of phenol and NADPH affected the Km values for phenol at 25 degrees C, but not at 10 degrees C. The results support the idea that the enzyme in situ is in a more favourable position for catalysis than the purified enzyme and that slow conformational changes, triggered by binding of phenol, become rate limiting above 10 degrees C.

Thiol- and pH-modulated slow conformational changes and cooperativity of phenol-binding sites in phenol hydroxylase.

Spectrophotometric titration of phenol hydroxylase (EC 1.14.13.7) with phenol indicated interacting sites for phenol binding. In the absence of added thiol, the cooperativity was positive up to a pH around 8.0 but negative at higher pH values. With added thiol-ethylenediaminetetraacetate, the cooperativity was negative at all investigated pH values. Conversely, a corresponding titration of an enzyme preparation that had been selectively modified in its two most reactive SH groups indicated positive cooperativity at all studied pH values. This selective modification affects the activity of the enzyme to a very minor degree, in contrast to more extensive SH blocking, which displaces flavin adenine dinucleotide with a corresponding loss of activity [Neujahr, H. Y., & Gaal, A. (1975) Eur. J. Biochem. 58, 351-357]. The reactivity of SH groups in the enzyme was significantly decreased after turnover. Thiol treatment restored it to that of the native enzyme. Adding phenol prior to reduced nicotinamide adenine dinucleotide phosphate (NADPH) in the assay of phenol hydroxylase gave immediate linearity and higher initial rates than when NADPH was added first. In the absence of added thiol, there was then a shift of the pH optimum. The results indicate slow conformational changes limiting the rate of the overall reaction. The two most reactive SH groups of phenol hydroxylase, though not participating in any obvious redox reactions, are important for these slow conformational changes and for the cooperativity of phenol-binding sites, wherein the anionic S- forms may be involved (pKa for cysteine is 8.35).

Induction of high-affinity phenol uptake in glycerol-grown Trichosporon cutaneum.

Two uptake systems for phenol are identified in Trichosporon cutaneum. One is an inducible, high-affinity system, sensitive to protonophores. It is induced coordinately with phenol hydroxylase but can operate independently of phenol metabolism. The other is a constitutive, low-affinity system with different specificity and different pH optimum. It is not sensitive to protonophores.

Phenol hydroxylase from yeast. A model for phenol binding and an improved purification procedure.

The binding of phenol to phenol hydroxylase was studied by equilibrium dialysis, spectrophotometric titration and by steady-state kinetics. A binding model with two identical, negatively cooperative, effector/substrate-binding sites per enzyme dimer is proposed. The spectral perturbation caused by phenol and the kinetics of the overall reaction were analysed with relation to the enzyme-phenol complexes of the binding model. The main part of the spectral perturbation as well as of the increase in NADPH oxidation rate was achieved by one molecule of phenol bound per enzyme dimer. The properties of different enzyme-phenol complexes, in terms of spectral changes, hydroxylase activity, oxidase activity and substrate inhibition are discussed. A new purification procedure is described.

Chemical modification of phenol hydroxylase by ethoxyformic anhydride.

Phenol hydroxylase was inactivated by ethoxyformic anhydride. Part of the inactivation was related to modification of histidyl residues. The remaining part of the inactivation is proposed to be due to the modification of a lysyl residue which, we suggest, is identical with the one previously described, being essential for the binding of NADPH [Neujahr, H. Y. and Kjellén, K. G. (1980) Biochemistry 19, 4967-4972]. The overall inactivation reaction is biphasic and follows pseudo-first-order kinetics. Numerical analysis of kinetic data was applied to discriminate between simultaneous reactions at different sites. It is proposed that phenol hydroxylase contains two essential histidyl residues, located in or near the NADPH-binding sites. Ethoxyformylation of the lysyl residue(s) caused tightening of the binding of phenol and perturbation of the FAD spectrum of phenol hydroxylase, similar to that caused by phenolic effectors.

In situ and in vitro kinetics of phenol hydroxylase.

The half saturation constant for phenol was much lower with phenol hydroxylase in situ than with the purified enzyme, whereas the constant for NADPH was higher. In both cases, the linearized plots of the Michaelis-Menten equation were biphasic and the half saturation constants for all phenolic substrates were several times lower, when the phenol was added to the assay medium before NADPH, than when NADPH was added first. There was a similar, but much smaller, effect on the half saturation constants for NADPH. The V-values were not affected by the order of addition. The results suggest slow conformational changes in the enzyme during the overall reaction, which seem even slower, when the enzyme is measured in situ.

Growth and enzyme synthesis during continuous culture of Trichosporon cutaneum on phenol.

The soil yeast Trichosporon cutaneum was grown in continuous culture with phenol as the sole carbon source. The cultures were operated as carbon-limited chemostats or as steady-state continuous cultures without carbon limitation. Selected comparative runs were also conducted on glucose or acetate as carbon source. In addition to growth parameters, the activities of several intracellular enzymes were determined, comprising those directly involved in the degradation of phenol as well as auxiliary enzymes required for the generation of reducing power. All enzymes were assayed in detergent-permea-bilized cells. Phenol was found to serve as an excellent carbon source, comparable to glucose or acetate. The utilization of phenol in T. cutaneum is very efficient as indicated by a low maintenance requirement (0.01 g phenol/g cells.h). The cell yields obtained were on the order of 0.8 g cells/g phenol. Although the phenol-limited chemostats were run with fully phenol-induced cells, a further increase in the activities of isocitrate DH(NADP(+)), maleate DH and the phenol-degrading enzymes occurred after transition to nonlimiting condition. Enzyme activities increased in parallel with increasing phenol levels in the effluent, as well as with increasing toxicity. The significance of this phenomenon is discussed. The significance of this phenomenon is discussed. This elevation in enzyme activities in not related to an increase in specific growth rate.

Transport and hydrolysis of disaccharides by Trichosporon cutaneum.

Trichosporon cutaneum is shown to utilize six disaccharides, cellobiose, maltose, lactose, sucrose, melibiose, and trehalose. T. cutaneum can thus be counted with the rather restricted group of yeasts (11 to 12% of all investigated) which can utilize lactose and melibiose. The half-saturation constants for uptake were 10 +/- 3 mM sucrose or lactose and 5 +/- 1 mM maltose, which is of the same order of magnitude as those reported for Saccharomyces cerevisiae. Our results indicate that maltose shares a common transport system with sucrose and that there may be some interaction between the uptake systems for lactose, cellobiose, and glucose. Lactose, cellobiose, and melibiose are hydrolyzed by cell wall-bound glycosidase(s), suggesting hydrolysis before or in connection with uptake. In contrast, maltose, sucrose, and trehalose seem to be taken up as such. The uptake of sucrose and lactose is dependent on a proton gradient across the cell membrane. In contrast, there were no indications of the involvement of gradients of H+, K+, or Na+ in the uptake of maltose. The uptake of lactose is to a large extent inducible, as is the corresponding glycosidase. Also the glycosidases for cellobiose, trehalose, and melibiose are inducible. In contrast, the uptake of sucrose and maltose and the corresponding glycosidases is constitutive.

Uptake of phenol by Trichosporon cutaneum.

The soil yeast Trichosporon cutaneum, which is distinguished by having a strictly oxidative metabolism, can be induced to utilize phenol as a sole carbon source. The present paper shows that such phenol-induced cells contain a specific, energy-dependent uptake system for phenol. Phenol uptake is not directly linked to its o-hydroxylation inside the cell, the first step of phenol metabolism. The Km for uptake is 235 +/- 30 microM, that for hydroxylation only 4.5 +/- 0.5 microM. Further, the phenol analog 2,6-dimethylphenol, which can not be hydroxylated, competes with phenol for the uptake system. The pH dependence of uptake indicates that phenolate is an essential form during the uptake process. The energy requirement for phenol uptake is indicated by effects of various inhibitors of energy generation, including proton-conducting uncouplers. Direct monitoring of proton movements in a pH-stat during phenol uptake indicates a phenol-proton symport. One proton is cotransported with every phenol molecule. Phenol competes with the uptake of sucrose and glycerol by cells grown on these substrates. Under such conditions the uptake of phenol seems to proceed through a different system, with lower affinity for phenol than in phenol-grown cells.