NMR studies on p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens and salicylate hydroxylase from Pseudomonas putida

p-Hydroxybenzoate hydroxylase from Pseudomonas fluorescens and salicylate hydroxylase from Pseudomonas putida have been reconstituted with 13C- and 15N-enriched FAD. The protein preparations were studied by 13C-NMR, 15N-NMR and 31P-NMR techniques in the oxidized and in the two-electron-reduced states. The chemical shift values are compared with those of free flavin in water or chloroform. It is shown that the pi electron distribution in oxidized free p-hydroxybenzoate hydroxylase is comparable to free flavin in water, and it is therefore suggested that the flavin ring is solvent accessible. Addition of substrate has a strong effect on several resonances, e.g. C2 and N5, which indicates that the flavin ring becomes shielded from solvent and also that a conformational change occurs involving the positive pole of an alpha-helix microdipole. In the reduced state, the flavin in p-hydroxybenzoate hydroxylase is bound in the anionic form, i.e. carrying a negative charge at N1. The flavin is bound in a more planar configuration than when free in solution. Upon binding of substrate the resonances of N1, C10a and N10 shift upfield. It is suggested that these upfield shifts are the result of a conformational change similar, but not identical, to the one observed in the oxidized state. The 13C chemical shifts of FAD bound to apo(salicylate hydroxylase) indicate that in the oxidized state the flavin ring is also fairly solvent accessible in the free enzyme. Addition of substrate has a strong effect on the hydrogen bond formed with O4 alpha. It is suggested that this is due to the exclusion of water from the active site by the binding of substrate. In the reduced state, the flavin is anionic. Addition of substrate forces the flavin ring to adopt a more planar configuration, i.e. a sp2-hybridized N5 atom and a slightly sp3-hybridized N10 atom. The NMR results are discussed in relation to the reaction catalyzed by the enzymes.     

Oxidation-reduction potential studies on p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens

The oxidation-reduction potential of p-hydroxybenzoate hydroxylase (4-hydroxybenzoate, NADPH: oxygen oxidoreductase (3-hydroxylating), EC 1.14.13.2) from Pseudomonas fluorescens has been measured in the presence and absence of p-hydroxybenzoate using spectrocoulometry. The native enzyme demonstrated a two-electron midpoint potential of -129 mV during the initial reductive titration. The midpoint potential observed during subsequent oxidative and reductive titrations was -152 mV. This marked hysteresis is proposed to arise from the oxidation and reduction of the known air-sensitive thiol group on the enzyme (Van Berkel, W.J.H. and Müller, F. (1987) Eur. J. Biochem. 167, 35-46). Redox titrations of the enzyme in the presence of substrate showed a two-electron midpoint potential of -177 mV. No spectral or electrochemical evidence for the thermodynamic stabilization of any flavin semiquinone was observed in the titrations performed. These data show that the affinity of the apoenzyme for the hydroquinone form of FAD is 150-fold greater than for the oxidized flavin and that the substrate is bound to the reduced enzyme with a 3-fold lower affinity than to the oxidized enzyme. These data are consistent with the view that the stimulatory effect of substrate binding on the rate of enzyme reduction by NADPH is due to the respective geometries of the bound FAD and NADPH rather than to a large perturbation of the oxidation-reduction potential of the bound flavin coenzyme.     

The quinohaemoprotein lupanine hydroxylase from Pseudomonas putida

Lupanine hydroxylase catalyses the first reaction in the catabolism of the alkaloid lupanine by Pseudomonas putida. It dehydrogenates the substrate, which can then be hydrated. It is a monomeric protein of M(r) 72,000 and contains a covalently bound haem and a molecule of PQQ. The gene for this enzyme has been cloned and sequenced and the derived protein sequence has a 26 amino acid signal sequence at the N-terminal for translocation of the protein to the periplasm. Many of the features seen in the sequence of lupanine hydroxylase are common with other quinoproteins including the W-motifs that are characteristic of the eight-bladed propeller structure of methanol dehydrogenase. However, the unusual disulfide bridge between adjacent cysteines that is present in some PQQ-containing enzymes is absent in lupanine hydroxylase. The C-terminal domain contains characteristics of a cytochrome c and overall the sequence shows similarities with that of the quinohaemoprotein, alcohol dehydrogenase from Comamonas testosteroni. The gene coding for lupanine hydroxylase has been successfully expressed in Escherichia coli and a procedure has been developed to renature and reactivate the enzyme, which was found to be associated with the inclusion bodies. Reactivation required addition of PQQ and was dependent on calcium ions.     

Bioproduction of p-hydroxybenzoate from renewable feedstock by solvent-tolerant Pseudomonas putida S12

Pseudomonas putida strain S12palB1 was constructed that produces p-hydroxybenzoate from renewable carbon sources via the central metabolite l-tyrosine. P. putida S12palB1 was based on the platform strain P. putida S12TPL3, which has an optimised carbon flux towards l-tyrosine. Phenylalanine ammonia lyase (Pal) was introduced for the conversion of l-tyrosine into p-coumarate, which is further converted into p-hydroxybenzoate by endogenous enzymes. p-Hydroxybenzoate hydroxylase (PobA) was inactivated to prevent the degradation of p-hydroxybenzoate. These modifications resulted in stable accumulation of p-hydroxybenzoate at a yield of 11% (C-molC-mol(-1)) on glucose or on glycerol in shake flask cultures. In a glycerol-limited fed-batch fermentation, a final p-hydroxybenzoate concentration of 12.9mM (1.8gl(-1)) was obtained, at a yield of 8.5% (C-molC-mol(-1)). A 2-fold increase of the specific p-hydroxybenzoate production rate (q(p)) was observed when l-tyrosine was supplied to a steady-state C-limited chemostat culture of P. putida S12palB1. This implied that l-tyrosine availability was the bottleneck for p-hydroxybenzoate production under these conditions. When p-coumarate was added instead, q(p) increased by a factor 4.7, indicating that Pal activity is the limiting factor when sufficient l-tyrosine is available. Thus, two major leads for further improvement of the p-hydroxybenzoate production by P. putida S12palB1 were identified.     

Molecular cloning of salicylate hydroxylase genes from Pseudomonas cepacia and Pseudomonas putida

The sal gene encoding Pseudomonas cepacia salicylate hydroxylase was cloned and the sal encoding Pseudomonas putida salicylate hydroxylase was subcloned into plasmid vector pRO2317 to generate recombinant plasmids pTK3 and pTK1, respectively. Both cloned genes were expressed in the host Pseudomonas aeruginosa PAO1. The parental strain can utilize catechol, a product of the salicylate hydroxylase-catalyzed reaction, but not salicylate as the sole carbon source for growth due to a natural deficiency of salicylate hydroxylase. The pTK1- or pTK3-transformed P. aeruginosa PAO1, however, can be grown on salicylate as the sole carbon source and exhibited activities for the cloned salicylate hydroxylase in crude cell lysates. In wild-type P. cepacia as well as in pTK1- or pTK3-transformed P. aeruginosa PAO1, the presence of glucose in addition to salicylate in media resulted in lower efficiencies of sal expression P. cepacia apparently can degrade salicylate via the meta cleavage pathway which, unlike the plasmid-encoded pathway in P. putida, appears to be encoded on chromosome. As revealed by DNA cross hybridizations, the P. cepacia hsd and ht genes showed significant homology with the corresponding plasmid-borne genes of P. putida but the P. cepacia sal was not homologous to the P. putida sal. Furthermore, polyclonal antibodies developed against purified P. cepacia salicylate hydroxylase inactivated the cloned P. cepacia salicylate hydroxylase but not the cloned P. putida salicylate hydroxylase in P. aeruginosa PAO1. It appears that P. cepacia and P. putida salicylate hydroxylases, being structurally distinct, were probably derived through convergent evolution.     

Purification, properties, and oxygen reactivity of p-hydroxybenzoate hydroxylase from Pseudomonas aeruginosa

The monooxygenase, p-hydroxybenzoate hydroxylase (4-hydroxybenzoate, NADPH:oxygen oxidoreductase (3-hydroxylating), EC 1.14.13.2) has been isolated and purified from Pseudomonas aeruginosa. The reaction catalysed is linked to the pathways for degradation of aromatic compounds by microorganisms. The enzyme has been quantitatively characterized in this paper for use in the mechanistic analysis of the protein by site-directed mutagenesis. This can be achieved when the results presented are used in combination with the information on the sequence and structure of the gene for this protein and the high-resolution crystallographic data for the protein from P. fluorescens. The protein is a dimer of identical sub-units in solution, and has one FAD per polypeptide with a monomeric molecular weight of 45,000. A full steady-state kinetic analysis was carried out at the optimum pH (8.0). A Vmax of 3750 min-1 at 25 degrees C was calculated, and the enzyme has a concerted-substitution mechanism, involving the substrates, NADPH, oxygen, and p-hydroxybenzoate. Extensive analyses of the reactions of reduced enzyme with oxygen were carried out. The quality of the data obtained confirmed the mechanisms of these reactions as proposed earlier by the authors for the enzyme from P. fluorescens. It was found that the amino acid residue differences between enzyme from P. fluorescence and aeruginosa do marginally change some observed transient state kinetic parameters, even though the structure of the enzyme shows they have no direct role in catalysis. Thus, transient state kinetic analysis is an excellent tool to examine the role of amino acid residues in catalysis.     

Physiological characterization of Pseudomonas putida DOT-T1E tolerance to p-hydroxybenzoate

Pseudomonas putida DOT-T1E was isolated as a toluene-tolerant strain. We show that it is also able to grow on high concentrations (up to 17 g/liter [123 mM]) of p-hydroxybenzoate (4HBA). Tolerance to this aromatic carboxylic acid (up to 30 g/liter [217 mM]) is improved by preexposing the cells to low 4HBA concentrations; the adaptation process is caused by the substrate itself rather than by products resulting from its metabolism. The mechanisms of 4HBA tolerance seem to involve increased rigidity of the cell membrane as a result of a decrease in the cis/trans ratio of unsaturated fatty acids. In addition, energy-dependent efflux systems seem to operate in the exclusion of 4HBA from the cell membranes.     

Engineering of histidine tail in the N-terminal region of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens

The pobA of Pseudomonas florescens IFO14160 encoding a p-hydroxybenzoate hydroxylase (PHBH) was cloned, sequenced, and over-expressed in Escherichia coli. To facilitate the purification of PHBH, a fully active, tagged enzyme was constructed by engineering a two-, three-, or six-histidine tail in the N-terminal region (H2-, H3-, or H6-PHBH), or a six-histidine tail in the C-terminal region of the PHBH. The six-histidine tail in the C-terminal region of the enzyme could not be expressed with activity, while the other polyhistidine tails in the N-terminal region of the enzyme were highly active. However, the resulting H6-PHBH could not be purified by Ni-NTA chromatography because the H6-PHBH was so strongly bound to the supports that it could be eluted only after a significant change in conditions. On the other hand, H2-PHBH did not bind tightly to the Ni-chelate support. H3-PHBH was purified from the crude extract in a single step by using the optimized length and location of the polyhistidine tail in the enzyme.     

The temperature and pH dependence of some properties of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens

The free and complexed flavoprotein, p-hydroxybenzoate hydroxylase, was studied by light-absorption, circular-dichroism and fluorescence techniques as a function of the pH. The following compounds served as ligands for the enzyme: p-hydroxybenzoate, p-fluorobenzoate, benzoate, p-aminobenzoate and tetrafluoro-p-hydroxybenzoate. Depending on the technique used, the various ligands exhibit pH-dependent physical properties and dissociation constants. The data can be fitted with pKa values in the range 7.7-7.9. It is suggested that this pKa value belongs to a tyrosine residue in the active center of the enzyme. This assignment is supported by published data and additional experiments.     

Rapid relaxation processes in p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens revealed by subnanosecond-resolved laser-induced fluorescence

Time-resolved fluorescence studies were carried out on the FAD bound to p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens. The transient fluorescence exhibits complex decay kinetics with at least a short lifetime component in the 50-500-ps time region and a longer one in the range 1.5-3.5 ns. The shorter-lifetime component has a larger contribution in the presence of substrate (p-hydroxybenzoate) or inhibitor (p-aminobenzoate). The quenching of the fluorescence is both static and dynamic in nature. The decay of fluorescence anisotropy shows that the FAD environment is both flexible and rigid. The FAD mobility can be enhanced by dilution of the enzyme, by raising the temperature, or by the binding of substrate or inhibitors. The anisotropy results are interpreted in part in terms of a monomer-dimer equilibrium, whereby the FAD in the monomer contains much more flexibility. The above-mentioned effects induce a shift of the equilibrium to the monomeric side. From a constrained parameter fitting the dissociation constant is estimated to be about 1 microM for the free enzyme and somewhat higher for the binary complexes between the enzyme and substrate or inhibitor. pH variation has only a slight effect on fluorescence or anisotropy decay parameters, while dimethylsulfoxide appears to promote dissociation into monomers by weakening hydrophobic interaction between the subunits. The results are discussed in the light of newly developed insights into the functional role of rapid structural fluctuations in enzyme catalysis.     

Toluene bioconversion to p-hydroxybenzoate by fed-batch cultures of recombinant Pseudomonas putida.

A microbial oxidation process for the production of p-hydroxybenzoate (HBA) from toluene is reported. The oxidation reaction was studied in fed-batch fermentations using a recombinant Pseudomonas putida grown on glutamate as the sole carbon and energy source with salicylate and IPTG induction of tmoABCDE, and pchCF and phbz pathway genes, respectively. An average volumetric HBA productivity of 13.4 mg HBA x L(-1) x h(-1) was obtained under rapid growth conditions (glutamate excess), giving an HBA titer of 132 mg x L(-1) after 9.8 h of fermentation. This corresponded to an average specific HBA productivity of 7.2 microg HBA (mg total protein)(-1) x h(-1). In contrast, maximum HBA titers of 35 mg HBA x L(-1) were achieved in 27 h in comparative studies employing glutumate limited fed-batch cultures. A specific productivity of 4.1 microg HBA (mg total protein)(-1) x h(-1) and volumetric productivity of 1.3 mg HBA x L(-1) x h(-1) were calculated for the growth-rate restricted cultures. The differences in HBA production between the two cultures could be correlated to the levels of specific toluene-4-monooxygenase (T4MO) polypeptides. T4MO catalyzes the rate-limiting step in the pathway. Using experimental data, the half-life value of TmoA was calculated to be approximately 28 h. Assuming linear, monomolecular decay of TmoA, a specific degradation constant of 0.025 x h(-1) was calculated, which placed the stability of recombinant TmoA in the range of relatively stable proteins, even in the absence of co-expression of tmoF, the terminal oxidoreductase subunit of T4MO.     

Substitution of Arg214 at the substrate-binding site of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens.

The gene encoding p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens was cloned in Escherichia coli to provide DNA for mutagenesis studies on the protein product. A plasmid containing a 1.65-kbp insert of P. fluorescens chromosomal DNA was obtained and its nucleotide sequence determined. The DNA-derived amino acid sequence agrees completely with the chemically determined amino acid sequence of the isolated protein. The enzyme is strongly expressed under influence of the vector-encoded lac promotor and is purified to homogeneity in a simple three-step procedure. The relation between substrate binding, the effector role of substrate and hydroxylation efficiency was studied by use of site-directed mutagenesis. Arg214, in ion-pair interaction with the carboxy moiety of p-hydroxybenzoate, was replaced with Lys, Gln and Ala, respectively. The affinity of the free enzymes for NADPH is unchanged, whereas the affinity for the aromatic substrate is strongly decreased. For enzymes Arg214-->Ala and Arg214-->Gln, the effector role of substrate is lost. For enzyme Arg214-->Lys, binding of p-hydroxybenzoate highly stimulates the rate of flavin reduction. In the presence of substrate or substrate analogues, the reduced enzyme Arg214-->Lys fails to stabilize the 4 alpha-hydroperoxyflavin intermediate, essential for efficient hydroxylation. Like the wild-type, enzyme Arg214-->Lys is susceptible to substrate inhibition. From spectral and kinetic results it is suggested that secondary binding of the substrate occurs at the re side of the flavin, where the nicotinamide moiety of NADPH is supposed to bind.     

Molecular cloning and sequencing of the phenol hydroxylase gene from Pseudomonas putida BH

A genomic library of the phenol-degrading bacterium Pseudomonas putida BH was constructed in the broad host range cosmid pVK100 and introduced into Escherichia coli HB101. One of the recombinant cosmids recovered from catechol- and/or 2-hydroxymuconic semialdehyde-accumulating clones, pS10–45, had a 19.6-kb insert fragment which allowed P. putida KT2440 to grow on phenol as a sole carbon and energy source. Subcloning and expression studies indicated that the phenol hydroxylase gene cluster (pheA) is located on a 6.1-kb SacI fragment. The results of DNA sequencing of the SacI fragment revealed that the pheA gene cluster encodes a multicomponent phenol hydroxylase.