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Hydroxylation of Indole by Laboratory-evolved 2-Hydroxybiphenyl 3-Monooxygenase

2002; Elsevier BV; Volume: 277; Issue: 37 Linguagem: Inglês

10.1074/jbc.m205621200

1083-351X

Andreas J. Meyer, Michael Wu ̈rsten, Andreas Schmid, Hans‐Peter E. Kohler, Bernard Witholt,

Microbial bioremediation and biosurfactants

Directed enzyme evolution of 2-hydroxybiphenyl 3-monooxygenase (HbpA; EC 1.14.13.44) from Pseudomonas azelaica HBP1 resulted in an enzyme variant (HbpAind) that hydroxylates indole and indole derivatives such as hydroxyindoles and 5-bromoindole. The wild-type protein does not catalyze these reactions. HbpAind contains amino acid substitutions D222V and V368A. The activity for indole hydroxylation was increased 18-fold in this variant. Concomitantly, theKd value for indole decreased from 1.5 mm to 78 μm. Investigation of the major reaction products of HbpAind with indole revealed hydroxylation at the carbons of the pyrrole ring of the substrate. Subsequent enzyme-independent condensation and oxidation of the reaction products led to the formation of indigo and indirubin. The activity of the HbpAind mutant monooxygenase for the natural substrate 2-hydroxybiphenyl was six times lower than that of the wild-type enzyme. In HbpAind, there was significantly increased uncoupling of NADH oxidation from 2-hydroxybiphenyl hydroxylation, which could be attributed to the substitution D222V. The position of Asp222 in HbpA, the chemical properties of this residue, and the effects of its substitution indicate that Asp222 is involved in substrate activation in HbpA. Directed enzyme evolution of 2-hydroxybiphenyl 3-monooxygenase (HbpA; EC 1.14.13.44) from Pseudomonas azelaica HBP1 resulted in an enzyme variant (HbpAind) that hydroxylates indole and indole derivatives such as hydroxyindoles and 5-bromoindole. The wild-type protein does not catalyze these reactions. HbpAind contains amino acid substitutions D222V and V368A. The activity for indole hydroxylation was increased 18-fold in this variant. Concomitantly, theKd value for indole decreased from 1.5 mm to 78 μm. Investigation of the major reaction products of HbpAind with indole revealed hydroxylation at the carbons of the pyrrole ring of the substrate. Subsequent enzyme-independent condensation and oxidation of the reaction products led to the formation of indigo and indirubin. The activity of the HbpAind mutant monooxygenase for the natural substrate 2-hydroxybiphenyl was six times lower than that of the wild-type enzyme. In HbpAind, there was significantly increased uncoupling of NADH oxidation from 2-hydroxybiphenyl hydroxylation, which could be attributed to the substitution D222V. The position of Asp222 in HbpA, the chemical properties of this residue, and the effects of its substitution indicate that Asp222 is involved in substrate activation in HbpA. high pressure liquid chromatography N,N-dimethylformamide mass spectroscopy Indole is produced from the aromatic amino acid tryptophan in tryptophanase-synthesizing bacteria such as Escherichia coli(1DeMoss R.D. Moser K. J. Bacteriol. 1969; 98: 167-171Crossref PubMed Google Scholar). Enzymes that oxygenate the indole pyrrole ring are easily detectable because the reaction products are unstable and form pigments. This observation was first made when the naphthalene oxidation genes were expressed in E. coli, which resulted in the biosynthesis of indigo (2Ensley B.D. Ratzkin B.J. Osslund T.D. Simon M.J. Wackett L.P. Gibson D.T. Science. 1983; 222: 167-169Crossref PubMed Scopus (452) Google Scholar). Based on these results and because of its importance as a dye, the biocatalytic production of indigo by naphthalene dioxygenase was for some time a major goal of the biotech industry (3Murdock D. Ensley B.D. Serdar C. Thalen M. Bio/Technology. 1993; 11: 381-386Crossref PubMed Scopus (139) Google Scholar, 4Bialy H. Nat. Biotechnol. 1997; 15: 110Crossref PubMed Scopus (31) Google Scholar). Naphthalene dioxygenase was soon found not to be the only enzyme capable of indole oxidation: several other oxygenases that accept indole as a substrate have been identified (5Bhushan B. Samanta S.K. Jain R.K. Lett. Appl. Microbiol. 2000; 31: 5-9Crossref PubMed Scopus (54) Google Scholar, 6Gillam E.M.J. Notley L.M. Cai H. de Voss J.J. Guengerich F.P. Biochemistry. 2000; 39: 13817-13824Crossref PubMed Scopus (247) Google Scholar, 7O'Connor K.E. Dobson A.D.W. Hartmans S. Appl. Environ. Microbiol. 1997; 63: 4287-4291Crossref PubMed Google Scholar, 8O'Connor K.E. Hartmans S. Biotechnol. Lett. 1998; 20: 219-223Crossref Scopus (55) Google Scholar, 9Panke S. Witholt B. Schmid A. Wubbolts M.G. Appl. Environ. Microbiol. 1998; 64: 2032-2043Crossref PubMed Google Scholar, 10Hart S. Koch K.R. Woods D.R. J. Gen. Microbiol. 1992; 138: 211-216Crossref PubMed Scopus (36) Google Scholar). These are either enzymes similar to naphthalene dioxygenase that activate oxygen with iron centers or members of the cytochrome P450 family (6Gillam E.M.J. Notley L.M. Cai H. de Voss J.J. Guengerich F.P. Biochemistry. 2000; 39: 13817-13824Crossref PubMed Scopus (247) Google Scholar, 11Ensley B.D. Gibson D.T. J. Bacteriol. 1983; 155: 505-511Crossref PubMed Google Scholar). Although flavin nucleotides may be involved in electron transfer from cofactors in these proteins, none is known to be a flavoprotein oxygenase. This situation has changed with our recent finding that flavosystem oxygenases can be modified to accept unnatural substrates. 2-Hydroxybiphenyl 3-monooxygenase (HbpA; EC 1.14.13.44) fromPseudomonas azelaica HBP1 is a flavoprotein aromatic hydroxylase that catalyzes the hydroxylation of a variety of 2-substituted phenols to the corresponding catechols (12Eppink M.H.M. Schreuder H.A. van Berkel W.J.H. Protein Sci. 1997; 6: 2454-2458Crossref PubMed Scopus (132) Google Scholar, 13Kohler H.-P.E. Kohler-Staub D. Focht D.D. Appl. Environ. Microbiol. 1988; 54: 2683-2688Crossref PubMed Google Scholar, 14Suske W.A. Held M. Schmid A. Fleischmann T. Wubbolts M.G. Kohler H.-P.E. J. Biol. Chem. 1997; 272: 24257-24265Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). The mechanism of HbpA has been extensively studied by spectroscopic techniques, which revealed that molecular oxygen is activated via the formation of a flavin (C4a)-hydroperoxide (15Suske W.A. van Berkel W.J.H. Kohler H.-P.E. J. Biol. Chem. 1999; 274: 33355-33365Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), a common intermediate in the reaction cycle of this enzyme family (16van Berkel W.J.H. Eppink M.H.M. van der Bolt F.J.T. Vervoort J. Rietjens I.M.C.M. Schreuder H.A. Stevenson K.J. Massey V. Williams C.H., Jr. Flavins and Flavoproteins. University of Calgary Press, Calgary, Canada1997: 305-314Google Scholar). HbpA has a broad substrate spectrum, but does not hydroxylate indole (13Kohler H.-P.E. Kohler-Staub D. Focht D.D. Appl. Environ. Microbiol. 1988; 54: 2683-2688Crossref PubMed Google Scholar, 17Held M. Suske W. Schmid A. Engesser K.-H. Kohler H.-P.E. Witholt B. Wubbolts M.G. J. Mol. Catal. B. 1998; 5: 87-93Crossref Scopus (64) Google Scholar). By directed enzyme evolution, we recently changed the substrate reactivity of HbpA toward 2-tert-butylphenol, a substrate that is not converted by the wild-type enzyme (18Meyer A. Schmid A. Held M. Westphal A.H. Ro¨thlisberger M. Kohler H.-P.E. van Berkel W.J.H. Witholt B. J. Biol. Chem. 2002; 277: 5575-5582Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 19Meyer, A., Held, M., Schmid, A., Kohler, H.-P. E., and Witholt, B. (2002) Biotechnol. Bioeng., in pressGoogle Scholar). As a side product of this work, we also obtained an HbpA variant (which we denoted HbpAind) with activity for the hydroxylation of indole. In this study, we report the characterization of HbpAindwith respect to its catalytic properties. Whereas previous work on indole-oxygenating enzymes was mainly aimed at the biotechnological production of indigo, we were especially interested in the formation of the by-product indirubin. Indirubin and its analogs have been identified as potent inhibitors of cyclin-dependent kinases (20Hoessel R. Leclerc S. Endicott J.A. Nobel M.E.M. Lawrie A. Tunnah P. Leost M. Damiens E. Marie D. Marko D. Niederberger E. Tang W. Eisenbrand G. Meijer L. Nat. Cell Biol. 1999; 1: 60-67Crossref PubMed Scopus (734) Google Scholar). The crystal structure of cyclin-dependent kinase-2 in complex with indirubin derivatives showed that indirubin binds to the kinase ATP-binding site. As a consequence, it inhibits the proliferation of a wide range of cells and belongs to a group of novel anticancer compounds that act on the cell cycle (21Buolamwini J.K. Curr. Pharm. Des. 2000; 6: 379-392Crossref PubMed Scopus (229) Google Scholar). Escherichia coli JM101 (22Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York1989Google Scholar) and the pUC18 plasmid (23Yanish-Perron C. Vieira J. Messing J. Gene (Amst.). 1985; 33: 103-109Crossref PubMed Scopus (11465) Google Scholar) were used throughout for cloning and expression of thehbpA gene. Alkaline phosphatase (EC 3.1.3.1) was purchased from Roche Molecular Biochemicals (Basel, Switzerland). Catalase (EC1.11.1.6) from beef liver and formate dehydrogenase (EC 1.2.1.2) fromCandida boidinii were obtained from Fluka AG (Buchs, Switzerland). 4- and 5-hydroxyindole were from ICN Biomedicals Inc. (Aurora, OH). Components for the complex medium were obtained from Difco. All other chemicals were of the purest available quality and obtained from Fluka AG. Directed evolution of HbpA was performed by error-prone PCR based on in vitro manganese mutagenesis as described earlier (18Meyer A. Schmid A. Held M. Westphal A.H. Ro¨thlisberger M. Kohler H.-P.E. van Berkel W.J.H. Witholt B. J. Biol. Chem. 2002; 277: 5575-5582Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). The mutant library was subsequently plated onto LB medium. Cells harboring enzymes with activity for the hydroxylation of indole formed deep blue colonies. The single mutant D222V (HbpAD222V) was constructed using the QuickChangeTM site-directed mutagenesis kit from Stratagene (La Jolla, CA). Synthesis of wild-type HbpA and HbpAind was done in recombinant E. coli JM101 using M9 mineral medium and glycerol as the carbon source (17Held M. Suske W. Schmid A. Engesser K.-H. Kohler H.-P.E. Witholt B. Wubbolts M.G. J. Mol. Catal. B. 1998; 5: 87-93Crossref Scopus (64) Google Scholar). After harvesting the cells, the proteins were purified according to the method described recently (18Meyer A. Schmid A. Held M. Westphal A.H. Ro¨thlisberger M. Kohler H.-P.E. van Berkel W.J.H. Witholt B. J. Biol. Chem. 2002; 277: 5575-5582Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). The activity of wild-type HbpA and HbpAind was determined by measuring substrate consumption and product formation with reverse-phase high pressure liquid chromatography (HPLC)1 as described elsewhere (24Kohler H.-P.E. Schmid A. van der Maarel M. J. Bacteriol. 1993; 175: 1621-1628Crossref PubMed Google Scholar). The assay contained 0.2 μm HbpA or variant protein, 0.3 mm NADH, 0.2 mm2-hydroxybiphenyl, and 20 mm air-saturated phosphate buffer (pH 7.5). The activity of recombinant E. coli JM101 for the formation of indigo was determined in 250-ml shaking flasks containing 50 ml of LB medium (22Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York1989Google Scholar). Cultures of E. coli JM101 cells harboring a pUC18 derivative encoding HbpA or HbpAind were inoculated toA450 = 0.1. The cultures were incubated at 30 °C and vigorously shaken. When the culture color turned olive, samples of 1.1 ml were taken. 100 μl of these were used to determine the cell dry weight at 450 nm (25Witholt B. J. Bacteriol. 1972; 109: 350-364Crossref PubMed Google Scholar). The remaining 1 ml was centrifuged, and the supernatant was carefully removed. Cell-associated indigo was extracted with N,N-dimethylformamide (DMF) and quantified at 610 nm (ε610 = 15,900 liters mol−1 cm−1) (8O'Connor K.E. Hartmans S. Biotechnol. Lett. 1998; 20: 219-223Crossref Scopus (55) Google Scholar). The activity for indole was determined using an assay with NADH regeneration by formate dehydrogenase from C. boidinii (Fig.1) (26Kula M.-R. Wandrey C. Methods Enzymol. 1987; 136: 9-21Crossref PubMed Scopus (104) Google Scholar). The assay contained 0.2 μm HbpA or variant, 0.25 units of formate dehydrogenase, 160 mm sodium formate, 10 units of catalase from beef liver, 0.3 mm NADH, and 2 mm indole in 1 ml of 50 mm sodium phosphate buffer (pH 7.5). The assay was stopped by the addition of 20 μl of 10% (v/v) perchloric acid, and the precipitated proteins were spun down. The pellet- and tube-associated indigo was extracted with DMF and spectrophotometrically quantified. Dissociation constants between the enzymes and substrates were determined by monitoring the absorption changes of the enzyme-bound FAD upon binding of substrate (27van Berkel W. Westphal A. Eschrich K. Eppink M. de Kok A. Eur. J. Biochem. 1992; 210: 411-419Crossref PubMed Scopus (58) Google Scholar). For this, 12 μm purified wild-type HbpA and HbpAind were titrated with known concentrations of 2-hydroxybiphenyl or indole, and the resulting spectra were recorded using a Varian Cary E1 UV-visible spectrophotometer. Plotting Δ absorbance at a specific wavelength allowed the calculation of the dissociation constants by weighted nonlinear regression analysis (Enzfitter, Elsevier-Biosoft, Cambridge, U. K.). Analysis of compounds formed during in vitro indigo assays was done by reverse-phase HPLC-MS (Hewlett Packard 1100 MSD). The compounds were separated on a Hypersil ODS column (5 μm, 4.5 × 125 mm) and detected with a diode array detector and a mass spectrometer. Acidified (0.1% formic acid) H2O (solvent A) and 50% methanol and 50% acetonitrile (solvent B) were applied as the mobile phase according to the following timetable: 0 to 8 min, 85:15 solvent A/solvent B, flow rate of 1 ml min−1; gradient to 10 min, to 65:35 solvent A/solvent B, flow rate of 2 ml min−1; to 15 min, 65:35 solvent A/solvent B, flow rate of 2 ml min−1. Standards for isatin, 4- and 5-hydroxyindole, 2-indolinone, and indole were commercially available. 3-Indoxyl was prepared by dephosphorylating 3-indoxyl phosphate with alkaline phosphatase under anaerobic conditions. HPLC-MS analysis of the formed pigments was done under isocratic conditions at a flow rate of 1 ml min−1 with 70% (v/v) methanol as the mobile phase for the pigments derived from indole and with 40% (v/v) methanol for those derived from 4- and 5-hydroxyindole. The formed pigments were analyzed by TLC using silica gel cards and either toluene/acetone (4:1) or chloroform/acetone (97:3) as the mobile phase (28Eaton R.W. Chapman P.J. J. Bacteriol. 1995; 177: 6983-6988Crossref PubMed Google Scholar). For ultrathin sectioning, cells were fixed in 2.5% glutaraldehyde for 60 min and subsequently washed with water and embedded in low-melting-point agarose. After fixation in 1% OsO4 for 60 min, the blocks were dehydrated with ethanol and acetone and embedded in Epon/Araldite (29Hess W.M. Stain Technol. 1966; 41: 27-35Crossref PubMed Scopus (55) Google Scholar). Sections cut from the Epon/Araldite preparation were contrasted with uranyl acetate and lead citrate. Freeze-fracturing was carried out following standard procedures using a Balzers BAF 300 apparatus. The specimen sandwiches were fractured at −150 °C and immediately replicated with platinum/carbon. All pictures were taken with a Philips EM301 electron microscope. We recently changed the substrate reactivity of 2-hydroxybiphenyl 3-monooxygenase (HbpA) from P. azelaica HBP1 by directed evolution (18Meyer A. Schmid A. Held M. Westphal A.H. Ro¨thlisberger M. Kohler H.-P.E. van Berkel W.J.H. Witholt B. J. Biol. Chem. 2002; 277: 5575-5582Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). This work led to a mutant monooxygenase with increased activity for the hydroxylation of indole. The HbpA variant was denoted HbpAind. E. coli JM101 cultures synthesizing HbpAind turned deep blue when grown overnight on LB medium. Electron microscopy revealed the extracellular accumulation of material, which, we believe, consists of the water-insoluble pigment. After centrifugation, the pigment was extracted from the pellet with DMF. It was authenticated as indigo by TLC with toluene/acetone (4:1) as the mobile phase and commercially available indigo as the standard. This analysis also revealed the presence of a major by-product. TheRF value of this red pigment corresponded to theRF value determined earlier for indirubin (28Eaton R.W. Chapman P.J. J. Bacteriol. 1995; 177: 6983-6988Crossref PubMed Google Scholar). Analysis by HPLC-MS with 70% (v/v) methanol as the mobile phase showed two prominent molecular ion (MH+) peaks at m/z263 with retention times of 3.7 and 4.2 min. The UV-visible spectra and the fragmentation patterns were compared with literature data (28Eaton R.W. Chapman P.J. J. Bacteriol. 1995; 177: 6983-6988Crossref PubMed Google Scholar, 30Fearon W.R. Boggust W.A. Biochem. J. 1950; 46: 62-67Crossref PubMed Google Scholar,31Laatsch, H., and Ludwig-Ko¨hn, H. (1986) Liebigs Ann. Chem. 1847–1853Google Scholar), which confirmed that these two compounds were indigo (3.7 min) and indirubin (4.2 min). The formation of indigo by recombinant E. coli JM101 cells growing on LB medium was quantified. Cultures expressing thehbpAind gene accumulated 150 μm indigo within 8 h, whereas cultures of the host synthesizing HbpA remained colorless (Fig. 2). Recombinant protein levels in both cultures were checked by SDS-PAGE, and HbpA levels were determined to be in the same range of ∼20% of total cell protein. When pigments were extracted with DMF from recombinant E. coli JM101 cultures, the extract had a deep blue color. The blue color disappeared upon storage at room temperature, and the solution turned red (Fig. 3). The red pigment was analyzed by UV-visible spectroscopy, HPLC-MS, and TLC. It was authenticated as indirubin by comparison of the obtained results with literature data (28Eaton R.W. Chapman P.J. J. Bacteriol. 1995; 177: 6983-6988Crossref PubMed Google Scholar, 30Fearon W.R. Boggust W.A. Biochem. J. 1950; 46: 62-67Crossref PubMed Google Scholar, 31Laatsch, H., and Ludwig-Ko¨hn, H. (1986) Liebigs Ann. Chem. 1847–1853Google Scholar). Buffering the pH at a value of 7 or acidification with 0.1% (v/v) 10 m hydrochloric acid stabilized the formed indigo, whereas the addition of 0.1% (v/v) 10m sodium hydroxide or heating accelerated the disappearance of the blue color. The mutant monooxygenase HbpAind differs from wild-type HbpA by two amino acids: Asp222 was substituted by valine, and Val368 was substituted by alanine. HbpAind was purified according to the procedure developed for the wild-type enzyme with a yield of ∼30%. Analytical size-exclusion chromatography showed that the mutant monooxygenase formed a tetramer, which is also the case for wild-type HbpA (14Suske W.A. Held M. Schmid A. Fleischmann T. Wubbolts M.G. Kohler H.-P.E. J. Biol. Chem. 1997; 272: 24257-24265Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). To identify the major reaction products of indole hydroxylation,in vitro indigo formation assays were performed. After 30 min, a sample was taken and immediately saturated with argon. The proteins were precipitated and separated by centrifugation. The pigments in the pellet were extracted with DMF and analyzed by TLC. They were identified as indigo and indirubin. Analysis of the aqueous phase by HPLC-MS revealed the presence of 3-hydroxyindole (indoxyl) and 2-indolinone (oxindole). When a sample was taken after 60 min of assay time, isatin was also detected (TableI).Table IMajor reaction products of indole hydroxylation by HbpAindThe major reaction products of in vitro indole assays were authenticated by reverse-phase HPLC-MS. The assays contained 0.2 μm HbpAind, 0.25 units of formate dehydrogenase, 160 mm sodium formate, 10 units of catalase, 0.3 mm NADH, and 2 mm indole in 1 ml of 50 mm sodium phosphate buffer (pH 7.5).1-a All compounds were compared with commercially obtained standards. Open table in a new tab The major reaction products of in vitro indole assays were authenticated by reverse-phase HPLC-MS. The assays contained 0.2 μm HbpAind, 0.25 units of formate dehydrogenase, 160 mm sodium formate, 10 units of catalase, 0.3 mm NADH, and 2 mm indole in 1 ml of 50 mm sodium phosphate buffer (pH 7.5). 1-a All compounds were compared with commercially obtained standards. To investigate the substrate range of the HbpAindmutant monooxygenase, in vitro assays were performed with 4- and 5-hydroxyindole and 5-bromoindole. For the variant enzyme, color formation could be observed with all substrates, whereas the control assays with the wild-type protein remained colorless. The pigment derived from 4-hydroxyindole was purple, that from 5-hydroxyindole was orange, and that from 5-bromoindole was pink. The polar reaction products of the assays with 4- and 5-hydroxyindole were analyzed by HPLC-MS. For this, the in vitro assay was stopped by the addition of perchloric acid, and the proteins were spun down. Mass peaks (MH+) at m/z 150 were detected in the supernatants from both reactions. This mass correlates with the single hydroxylated substrates. The corresponding compounds eluted between 4.5 and 6.5 min when using 40% (v/v) methanol as the mobile phase. The main condensation products had a prominent molecular ion peak (MH+) at m/z 279 and had retention times of 4.1 min for the assay with 4-hydroxyindole and 3.5 min for the assay with the 5-substituted isomer. UV-visible spectra showed the peak at 4.1 min to have a maximum at 494 nm, whereas the peak at 3.5 min had a maximum at 480 nm. Thus, the molecular mass and the spectral properties indicated that the dihydroxy derivatives of indoxyl red were formed (see Fig. 7). Thein vitro activity of the purified proteins for the natural substrate 2-hydroxybiphenyl was determined by measuring substrate consumption and product formation by reverse-phase HPLC. Thekcat of HbpAind was significantly lower than that of the wild-type enzyme (TableII). Indole hydroxylation activities were determined in assays with purified proteins and NADH regeneration by formate dehydrogenase from C. boidinii. The assay mixture containing the mutant monooxygenase showed a blue color within the first 30 min, whereas the assay mixture with the wild-type enzyme remained white. HbpAind formed up to 170 μmindigo, whereas hardly any indigo formation could be observed for HbpA (Fig. 4). The indole hydroxylation activity of HbpAind was ∼20 milliunits mg−1purified protein or ∼18 times higher than the corresponding value for the wild-type enzyme.Table IIKd and kcat values of wild-type HbpA and HbpAind for 2-hydroxybiphenyl and indoleEnzyme2-HydroxybiphenylIndolekcat 2-aDetermined by measuring substrate consumption and product formation by reverse-phase HPLC. Values are the average of three independent measurements and have a standard error <10%.Kdkcat/Kdkcat 2-bDetermined by in vitro indigo formation assay with NADH regeneration by formate dehydrogenase. Values are the average of two independent measurements and have a standard error <10%.Kdkcat/Kds−1μms−1m−1s−1μms−1m−1HbpA15.69.9 ± 0.72-cValue adapted from Suske et al. (15).1.6 × 1065 × 10−31500 ± 703.3HbpAind2.38.8 ± 0.72.6 × 1059 × 10−278 ± 71.1 × 1032-a Determined by measuring substrate consumption and product formation by reverse-phase HPLC. Values are the average of three independent measurements and have a standard error <10%.2-b Determined by in vitro indigo formation assay with NADH regeneration by formate dehydrogenase. Values are the average of two independent measurements and have a standard error <10%.2-c Value adapted from Suske et al. (15Suske W.A. van Berkel W.J.H. Kohler H.-P.E. J. Biol. Chem. 1999; 274: 33355-33365Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Open table in a new tab The affinities of the enzymes for 2-hydroxybiphenyl and indole were determined by titration of the purified proteins with known concentrations of substrate (Fig.5, insets). Plotting the absorption difference at a specific wavelength as a function of substrate concentration (Fig. 5) allowed the determination of theKd values. Whereas the dissociation constants for 2-hydroxybiphenyl were in the same range for both proteins, theKd value for indole was 20-fold lower for HbpAind than for HbpA (Table II). Wild-type HbpA shows an uncoupling of NADH oxidation from 2-hydroxybiphenyl hydroxylation of 21% (18Meyer A. Schmid A. Held M. Westphal A.H. Ro¨thlisberger M. Kohler H.-P.E. van Berkel W.J.H. Witholt B. J. Biol. Chem. 2002; 277: 5575-5582Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Substitution of a single amino acid (V368A in HbpAT1) completely coupled these two reactions (18Meyer A. Schmid A. Held M. Westphal A.H. Ro¨thlisberger M. Kohler H.-P.E. van Berkel W.J.H. Witholt B. J. Biol. Chem. 2002; 277: 5575-5582Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). In contrast, there was significant uncoupling of NADH oxidation from hydroxylation for HbpAind compared with HbpA and HbpAT1 (TableIII). To investigate whether the increased uncoupling in HbpAind is an effect of the combination of the two amino acid substitutions or only due to the D222V exchange, the single mutant D222V (HbpAD222V) was constructed by site-directed mutagenesis. Uncoupling of NADH oxidation from 2-hydroxybiphenyl hydroxylation was 3-fold higher for the HbpAD222V mutant monooxygenase than for the wild-type protein (Table III).Table IIIUncoupling of NADH oxidation from 2-hydroxybiphenyl hydroxylationEnzymeAmino acid substitutionSpecific activityUnproductive NADH oxidationUncouplingNADH oxidation3-aDetermined by monitoring the NADH decrease at 340 nm.Product formation3-bDetermined by measuring substrate consumption and product formation by reverse-phase HPLC.s−1s−1s−1%HbpA3-cValues adapted from Meyer et al. (18).15.612.33.321HbpAT1 3-cValues adapted from Meyer et al. (18).V368A16.215.80.43HbpAD222VD222V15.05.19.966HbpAindD222V/V368A5.22.32.956The assays were performed at 30 °C in 20 mm sodium phosphate buffer (pH 7.5) containing 0.3 mm NADH and 0.2 mm 2-hydroxybiphenyl.3-a Determined by monitoring the NADH decrease at 340 nm.3-b Determined by measuring substrate consumption and product formation by reverse-phase HPLC.3-c Values adapted from Meyer et al. (18Meyer A. Schmid A. Held M. Westphal A.H. Ro¨thlisberger M. Kohler H.-P.E. van Berkel W.J.H. Witholt B. J. Biol. Chem. 2002; 277: 5575-5582Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Open table in a new tab The assays were performed at 30 °C in 20 mm sodium phosphate buffer (pH 7.5) containing 0.3 mm NADH and 0.2 mm 2-hydroxybiphenyl. Indole is oxidized by different oxygenases that contain either protein-bound iron or cytochrome to activate molecular oxygen (6Gillam E.M.J. Notley L.M. Cai H. de Voss J.J. Guengerich F.P. Biochemistry. 2000; 39: 13817-13824Crossref PubMed Scopus (247) Google Scholar, 32Carredano E. Karlsson A. Kauppi B. Choudhury D. Parales R.E. Parales J.V. Lee K. Gibson D.T. Eklund H. Ramaswamy S. J. Mol. Biol. 2000; 296: 701-712Crossref PubMed Scopus (129) Google Scholar). No flavoprotein oxygenase has thus far been shown to accept indole as a substrate. A 2-hydroxybiphenyl 3-monooxygenase variant (HbpAind), which we obtained during directed evolution of HbpA, showed the ability to hydroxylate indole. Thekcat/Kd of HbpAind for indole was 330-fold higher than that of wild-type HbpA and is in the same order as the catalytic efficiency determined for the engineered fatty-acid hydroxylase P450 BM-3 (33Li Q.-S. Schwaneberg U. Fischer M. Schmid R.D. Chem. Eur. J. 2000; 6: 1531-1536Crossref PubMed Scopus (169) Google Scholar). This P450 variant was obtained by saturation mutagenesis and is the enzyme with the highest known catalytic efficiency for indole. Cultures of E. coli JM101 cells that synthesized HbpAind during growth on LB medium had an indigo productivity of ∼5 mg liter−1 h−1. In comparison, a recombinant E. coli HB101 culture that expressed the naphthalene dioxygenase genes produced ∼1 mg liter−1 h−1 during growth on the same medium to a similar cell density (2Ensley B.D. Ratzkin B.J. Osslund T.D. Simon M.J. Wackett L.P. Gibson D.T. Science. 1983; 222: 167-169Crossref PubMed Scopus (452) Google Scholar). E. coli cultures that synthesized human P450 cytochromes reached productivities of ∼0.3 mg liter−1 h−1 on fortified terrific broth (TB) medium (6Gillam E.M.J. Notley L.M. Cai H. de Voss J.J. Guengerich F.P. Biochemistry. 2000; 39: 13817-13824Crossref PubMed Scopus (247) Google Scholar). Thus, the HbpAind flavoprotein recombinant is considerably (5–15-fold) more active in vivoin the formation of indigo from complex medium. Analysis of the pigments formed also showed the presence of the by-product indirubin, a structural isomer of indigo that is known to be formed by indole-oxidizing enzymes (4Bialy H. Nat. Biotechnol. 1997; 15: 110Crossref PubMed Scopus (31) Google Scholar, 10Hart S. Koch K.R. Woods D.R. J. Gen. Microbiol. 1992; 138: 211-216Crossref PubMed Scopus (36) Google Scholar). Indigo extracted from recombinant E. coli cultures showed only limited stability when stored in DMF at room temperature. This could be attributed to the pH of the solution. It is known from denim manufacturing that at basic pH indigo is chemically reduced to its water-soluble form, indigo white. In contrast, the formed indirubin was stable when extracted and stored in DMF. The products of indole oxidation by different mono- and dioxygenases have been investigated. Indole epoxide has been suggested as a product of the reaction catalyzed by styrene monooxygenase (7O'Connor K.E. Dobson A.D.W. Hartmans S. Appl. Environ. Microbiol. 1997; 63: 4287-4291Crossref PubMed Google Scholar), and indoxyl (3-hydroxyindole) has been identified as a hydroxylation product of cytochrome P450 enzymes (6Gillam E.M.J. Notley L.M. Cai H. de Voss J.J. Guengerich F.P. Biochemistry. 2000; 39: 13817-13824Crossref PubMed Scopus (247) Google Scholar). Oxidation by naphthalene dioxygenase results in the formation of 2,3-dihydroxy-2,3-dihydroindole (2Ensley B.D. Ratzkin B.J. Osslund T.D. Simon M.J. Wackett L.P. Gibson D.T. Science. 1983; 222: 167-169Crossref PubMed Scopus (452) Google Scholar). All these reaction products are unstable and spontaneously form pigments. The identification of the intermediates formed during in vitro indigo assays with HbpAind suggests a similar route as observed for the P450 enzymes (Fig.6). The identification of 3-hydroxyindole and 2-indolinone indicates direct hydroxylation at the carbons of the pyrrole ring of indole. In contrast to the P450 enzymes, no hydroxylation at the benzene ring of the substrate was observed (6Gillam E.M.J. Notley L.M. Cai H. de Voss J.J. Guengerich F.P. Biochemistry. 2000; 39: 13817-13824Crossref PubMed Scopus (247) Google Scholar). The presence of isatin could have two origins: it was produced either by hydroxylation of indoxyl or oxindole or by the decomposition of indigo and indirubin. The formation of indigo and indirubin from the enzymatic hydroxylation products of indole is spontaneous and biocatalyst-independent. In short, condensation of two molecules of indoxyl followed by air oxidation leads to the production of indigo, whereas condensation of indoxyl and 2-indolinone yields indirubin (28Eaton R.W. Chapman P.J. J. Bacteriol. 1995; 177: 6983-6988Crossref PubMed Google Scholar,34Russell G.A. Kaupp G. J. Am. Chem. Soc. 1969; 91: 3851-3859Crossref Scopus (113) Google Scholar). In addition, indirubin can also be formed by the reaction of indoxyl with isatin (4Bialy H. Nat. Biotechnol. 1997; 15: 110Crossref PubMed Scopus (31) Google Scholar, 35Maugard T. Enaud E. Choisy P. Legoy M.D. Phytochemistry. 2001; 58: 897-904Crossref PubMed Scopus (157) Google Scholar). That this latter reaction does indeed take place in the case of indirubin formation by HbpAind is supported by the fact that the ratio of indirubin to indigo increased with time when recombinant E. coli cells were grown on LB medium. Indirubin inhibits cyclin-dependent kinases and therefore belongs to a group of promising anticancer compounds (20Hoessel R. Leclerc S. Endicott J.A. Nobel M.E.M. Lawrie A. Tunnah P. Leost M. Damiens E. Marie D. Marko D. Niederberger E. Tang W. Eisenbrand G. Meijer L. Nat. Cell Biol. 1999; 1: 60-67Crossref PubMed Scopus (734) Google Scholar). Analogs such as indirubin 3′-monoxime and halogenated indirubins show even higher potency (36Marko D. Schatzle S. Friedel A. Genzlinger A. Zankl H. Meijer L. Eisenbrand G. Br. J. Cancer. 2001; 84: 283-289Crossref PubMed Scopus (174) Google Scholar). This increased biological activity can be attributed to the lower hydrophobicity of the derivatives compared to indirubin. Thus, the uptake of the compound is facilitated and the probability that it reaches the biological sites of action is increased (37Hansch C. Acc. Chem. Res. 1969; 2: 232-239Crossref Scopus (878) Google Scholar). In this context, we investigated the substrate spectrum of HbpAind. The variant showed activity with several indole derivatives such as 4- and 5-hydroxyindole. The products of these reactions are potentially interesting because their log P values are significantly different from that of the unsubstituted indirubin and are hardly accessible by chemical means. However, analysis of the formed pigments showed that the dihydroxyindirubin derivatives were only a minor product of the reaction of HbpAind with hydroxyindoles. The mass and the spectral properties of the major condensation product indicated that mainly the indoxyl red derivatives were formed. Indoxyl red is known to be formed from the reaction of 3-oxo-3H-indole with indole (38Capdevielle P. Maumy M. Tetrahedron Lett. 1993; 34: 2953-2956Crossref Scopus (25) Google Scholar). The proposed pathway for the formation of the dihydroxy derivatives by HbpAind from hydroxyindole is shown in Fig. 7. The electron-donating hydroxyl group at the benzene ring facilitates the oxidation of the indolinone compared with the unsubstituted compound and may explain why indoxyl red was not detected from the reaction of HbpAind with indole. Indoxyl red shares a high degree of structural similarities with indirubin. The dihydroxy derivatives have a strongly decreased hydrophobicity, and their potency in inhibiting cyclin-dependent kinases is worth investigating. We characterized HbpAind with respect to its catalytic properties. The variant’s activity for indole was increased by ∼18-fold compared with the wild-type enzyme. This increase was concomitant with an enhanced affinity of the enzyme for this substrate. The in vitro activity of the mutant monooxygenase for the natural substrate 2-hydroxybiphenyl was significantly decreased, whereas its affinity for this substrate remained unchanged. This is mostly due to a slower flavin reduction, as indicated by the reduced NADH oxidation rate. In addition, uncoupling of NADH oxidation from substrate hydroxylation was significantly increased in HbpAind. HbpAind evolved from the single mutant V368A (HbpAT1) by directed enzyme evolution. In contrast to wild-type HbpA, HbpAT1 fully couples NADH oxidation to 2-hydroxybiphenyl hydroxylation. We have suggested that this is due to the stabilization and/or improved positioning of the flavin (C4a)-hydroperoxide toward the substrate (18Meyer A. Schmid A. Held M. Westphal A.H. Ro¨thlisberger M. Kohler H.-P.E. van Berkel W.J.H. Witholt B. J. Biol. Chem. 2002; 277: 5575-5582Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). This enhanced hydroxylation efficiency was completely destroyed by the D222V substitution: in HbpAind, the uncoupling was twice that of the wild-type protein with 2-hydroxybiphenyl as substrate, whereas the unproductive NADH oxidation rate was similar in both enzymes. The single mutant HbpAD222V even showed a 3-fold increased uncoupling, which was concomitant with a 3-fold increased unproductive NADH oxidation rate. Thus, the substitution D222V in HbpAind and HbpAD222V directly increased the ratio of flavin (C4a)-hydroperoxide decay to 2,3-dihydroxybiphenyl formation (Fig. 8). This ratio is primarily influenced by stabilization of the flavin (C4a)-hydroperoxide, solvent access to the active site, and the reactivity of the substrate toward electrophilic attack by the terminal oxygen of the peroxide. According to the structural model of HbpA (18Meyer A. Schmid A. Held M. Westphal A.H. Ro¨thlisberger M. Kohler H.-P.E. van Berkel W.J.H. Witholt B. J. Biol. Chem. 2002; 277: 5575-5582Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), Asp222 is located close to the bound substrate in the substrate-binding domain. Interestingly, Asp222 of HbpA corresponds to Tyr201 of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens (39Gatti D.L. Palfey B.A. Lah M.S. Entsch B. Massey V. Ballou D.P. Ludwig M.L. Science. 1994; 266: 110-114Crossref PubMed Scopus (177) Google Scholar, 40Schreuder H.A. Mattevi A. Obmolova G. Kalk K.H. Hol W.G.J. van der Bolt F.J.T. van Berkel W.J.H. Biochemistry. 1994; 33: 10161-10170Crossref PubMed Scopus (103) Google Scholar). Inp-hydroxybenzoate hydroxylase, Tyr201 is critically involved in the ionization of the substrate, thus affecting flavin movement and allowing efficient hydroxylation (41Palfey B.A. Moran G.R. Entsch B. Ballou D.B. Massey V. Biochemistry. 1999; 38: 1153-1158Crossref PubMed Scopus (86) Google Scholar, 42Gatti D.L. Entsch B. Ballou D.P. Ludwig M.L. Biochemistry. 1996; 35: 567-578Crossref PubMed Scopus (46) Google Scholar). The substitution Y201F results in an increased uncoupling of NADH oxidation from substrate hydroxylation up to 95% (43Entsch B. Palfey B.A. Ballou D.P. Massey V. J. Biol. Chem. 1991; 266: 17341-17349Abstract Full Text PDF PubMed Google Scholar). In phenol 2-monooxygenase from Trichosporon cutaneum, Tyr289 is hydrogen-bonded with the hydroxyl group of the substrate, comparable to Tyr201 in p-hydroxybenzoate hydroxylase (44Enroth C. Neujahr H. Schneider G. Lindqvist Y. Structure. 1998; 6: 605-617Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Substitution of this residue by Phe increased the uncoupling from 10 to 34% (45Xu D. Ballou D.P. Massey V. Biochemistry. 2001; 40: 12369-12378Crossref PubMed Scopus (38) Google Scholar). With respect to the localization of Asp222 in the HbpA model, the chemical properties of this residue, and the effects resulting from its substitution, it is likely that Asp222 plays a similar role in HbpA as do the tyrosine residues in p-hydroxybenzoate hydroxylase and phenol 2-monooxygenase. In summary, we characterized the HbpAind mutant monooxygenase, the first flavoprotein able to hydroxylate indole. These investigations point to the importance of Asp222 in the catalytic cycle of HbpA. Thus, the results obtained here may serve as the basis for further elucidation of the mechanism of substrate activation in this enzyme. We thank Dr. E. Wehrli (ETH Zurich) for performing the electron microscopy.