Homogenization and crystallization of histidine ammonia-lyase by exchange of a surface cysteine residue

Torsten F. Schwede1, Mathias Bädeker1, Martin Langer2, Janos Rétey2 and Georg E. Schulz1,3

1 Institut für Organische Chemie und Biochemie, Albertstrasse 21,D-79104 Freiburg im Breisgau and 2 Institut für Organische Chemie, Lehrstuhl Biochemie der Universität, Richard-Willstätter-Allee, D-76128 Karlsruhe, Germany


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Histidase (histidine ammonia-lyase, EC 4.3.1.3) from Pseudomonas putida was expressed in Escherichia coli and purified. In the absence of thiols the tetrameric enzyme gave rise to undefined aggregates and suitable crystals could not be obtained. The solvent accessibility along the chain was predicted from the amino acid sequence. Among the seven cysteines, only one was labeled as `solvent-exposed'. The exchange of this cysteine to alanine abolished all undefined aggregations and yielded readily crystals diffracting to 1.8 Å resolution.

Keywords: disulfide engineering/Ellman's reaction/histidine ammonia-lyase/unspecific aggregation/X-ray crystallography


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Histidine degradation to L-glutamate and ammonia starts with the non-oxidative elimination of the amino group catalyzed by histidase (histidine ammonia-lyase, EC 4.3.1.3) (Figure 1Go). Deficiency of histidase in humans is known as histidinemia (Taylor et al., 1991Go). Histidases are homologous (about 25% sequence identity) to phenylalanine ammonia-lyases that are important enzymes of the plant secondary metabolism and potential herbicide targets (Hahlbrock and Scheel, 1989Go). Both enzymes follow the same reaction mechanism (Langer et al., 1995Go; Schuster and Rétey, 1995Go) using a putative dehydroalanine which is formed autocatalytically from a serine residue, i.e. Ser143 in Pseudomonas putida histidase. The mechanism is considered as the only known enzymatic analogue of Friedel–Crafts reactions (for a review, see Rétey, 1996Go). Histidase from P.putida was the first enzyme of this family to be cloned and purified to homogeneity. The enzyme is a homotetramer of 509 amino acid residues (Mr = 53 559) per subunit and shows an unusually high melting temperature of 83°C for an enzyme isolated from a mesophilic organism (Hernandez and Phillips, 1993Go; Langer et al., 1994Go).



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Fig. 1. Non-oxidative elimination of the amino group of histidine as catalyzed by histidine ammonia-lyase.

 
Histidase has been reported to exist in diverse isoforms, which differ with respect to oxidation state and binding of divalent cations such as Mg2+ (Klee, 1972Go; see Figure 2Go in Langer et al., 1994Go). In agreement with previous data (Klee, 1970Go), gel permeation chromatography revealed undefined aggregations of the tetrameric protein (Figure 2AGo). These were completely reversible upon addition of reducing agents such as 10 mM dithiothreitol (DTT). Unfortunately, the presence of reducing agents did not suffice for producing suitable crystals. Here, we report the identification and exchange of a cysteine residue at the protein surface that resulted in monodisperse protein solutions and yielded readily high-quality crystals.



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Fig. 2. Characterization of histidase. (A) Analysis of the oligomeric state of wild-type (dotted curve) and mutant Cys273->Ala (solid curve) by gel permeation chromatography under non-reducing conditions. (B) Determination of sulfhydryl groups using the method of Ellman (1959). After 7 h of incubation we find the seven cysteine residues of the DNA-derived sequence of wild-type histidase ({circ} and {square}) and the six cysteines of mutant Cys273->Ala (+ and x).

 

    Materials and methods
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 Abstract
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 Materials and methods
 Results and discussion
 References
 
Expression, purification and crystallization of the wild-type enzyme

The histidase gene from P.putida was expressed in Escherichia coli BL21(DE3-pT7-7 hutH) cells following Langer et al. (1994). Purification was performed by ammonium sulfate fractionation (2.1–3.0 M), gel permeation chromatography (Superdex 200, 26/60) and ion-exchange chromatography (Q-Sepharose FF) using throughout a buffer of 50 mM KH2PO4, pH 7.2, and 1 mM MgSO4. Typically 120–180 mg of pure histidase were obtained from a 1 l culture. Crystallization conditions were initially found by screening in hanging drops (Jancarik and Kim, 1991Go). Crystallization occurred only at acidic pH and was accompanied by protein precipitation (Teo et al., 1998Go). The elaborated droplet composition was a 1:1 mixture of a 12 mg/ml protein solution with the reservoir buffer (1.6 M sodium potassium phosphate, 0.1 M HEPES, 1.5 mM EDTA, 3% dioxane, pH 3.85).

Protein characterization

The protein melting temperature was determined by monitoring the tryptophan fluorescence (Hitachi Perkin-Elmer MPF-2A, {lambda}ex = 282 nm, {lambda}em = 334 nm) during thermal denaturation (rate 1°C/min). The oligomeric state was determined by gel permeation chromatography (Superdex 200, 16/60). Thiol content was determined using a modification of the method of Ellman (1959). After complete denaturation and reduction for 30 min at 75°C (8 M urea, 10 mM DTT, 150 mM NaCl, 1 mM EDTA, 20 mM K2HPO4, pH 8.0), excess DTT was removed by multiple ultrafiltration (Centricon 30, Millipore). The protein concentration was determined spectrophotometrically and the reaction of 5,5'-dithiobis-2-nitrobenzoate with sulfhydryl groups was monitored at {lambda} = 412 nm ({varepsilon}TNB = 13 600 M–1 cm–1) and scaled on a molar basis. Blinds were subtracted; all handling was carried out in the dark.

Mutagenesis and preliminary X-ray analysis

The gene of histidase was subcloned into M13mp18/19 in two fragments using an internal SalI restriction site (Langer et al., 1994Go). Site directed mutagenesis was performed using the Eckstein method (Sayers et al., 1988Go) with the primer 5'-GCACAAGAACGCCGACAAGGTCC-3'. The new crystallization conditions for the mutant Cys273->Ala were a 1:1 mixture of 14 mg/ml protein with the reservoir [2.0 M (NH4)2SO4, 1% glycerol, 2% PEG-400, 0.1 M HEPES, pH 8.1] as sitting drops.

X-ray diffraction data were collected at room temperature on a 30 cm MAR-Research imaging plate and processed with the program MOSFLM (CCP4, 1994Go). Data from mutant Cys273->Ala were collected using synchrotron radiation at beamline X11 ({lambda} = 0.907 Å, EMBL Outstation at DESY, Hamburg). Self-rotation functions were calculated with the program POLARRFN (CCP4, 1994Go) including data in the range between 10 and 5 Å. A heavy atom derivative was produced by adding 20 mM methylmercury acetate to the crystals in the droplet. The derivative data were collected with Cu K{alpha} radiation from a rotating anode source (RU200B, Rigaku).


    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Solutions of wild-type histidase gave rise to unspecific aggregates in the absence of reducing agents (Figure 2AGo). Using these heterogeneous solutions, we obtained erratically crystals that diffracted to about 4 Å resolution, but were not suitable for X-ray analysis. The addition of various reducing agents failed to improve the situation. The unit cell parameters were a = 89.1 Å, b = 137.6 Å, c = 163.4 Å, {alpha} {approx} ß {approx} {gamma} {approx} 90°. The space group could not be determined. We therefore decided to identify the cysteines responsible for the obstructing aggregation by prediction methods and to replace them. First, we determined the probability of each of the seven cysteine residues of the DNA-derived sequence of histidase (Consevage and Phillips, 1990Go) being located at the surface (Rost and Sander, 1994Go; Rost, 1996Go). The prediction labeled Cys273 as `exposed' and Cys221, Cys255 and Cys446 as `buried'. The remaining Cys41, Cys284 and Cys292 were not classified.

Consequently, we produced mutant Cys273->Ala without the `exposed' cysteine and checked it for its thiol content using a modified version of the method of Ellman (1959). The removal of one of the cysteines could be confirmed reproducibly (Figure 2BGo). Note that the exact values of the Ellman reaction were obtained only after about 7 h. In gel permeation chromatography (Figure 2AGo), the protein ran in a single peak, indicating that there are no higher aggregates. The melting temperature was not affected by the mutation. The specific activity (Langer et al., 1994Go) dropped to 18% of the wild-type activity (31.4 U/mg) indicating that Cys273 influences catalysis.

Mutant Cys273->Ala yielded readily rectangular crystals with average dimensions of 300x300x800 µm3 that diffracted isotropically to 1.8 Å resolution. Similar crystallization improvements in conjunction with protein homogenization have been reported for HIV-1 integrase by Jenkins et al. (1995), who had to produce 28 mutants, however, before they succeeded. The crystals belong to space group P21; the unit cell parameters are given in Table IGo. The unit cell contains most likely one histidase tetramer per asymmetric unit with a solvent content of 57%. A self-rotation function yielded strong peaks in the section {kappa} = 180° at ({omega}, {phi})-values of (0.9°, 0°) and (90.9°, 0°). These amounted to about 99% of those at the crystallographic twofold screw axis at ({omega}, {phi}) = (90°, 90°). This indicates that the tetramer has D2 symmetry and that one of its twofold axes runs nearly parallel to the crystallographic twofold screw axis. Diffraction data for a methylmercury acetate derivative were collected (Table IGo) and four major heavy atom sites were located in difference-Patterson maps using the program SHELXS-96 (Sheldrick et al., 1993Go). Further minor sites were identified in difference-Fourier maps. Given the extensive non-crystallographic symmetry, we expect that a single derivative will suffice for phasing.


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Table I. X-ray diffraction data
 
Conclusion

Cysteine residues at the surface of oligomeric proteins can cause unspecific aggregations and thereby complicate all handling and virtually inhibit the formation of highly ordered crystals. It has been shown that the identification of such residues by predicting solvent accessibility and their removal by site-directed mutagenesis is a powerful tool for improving protein homogeneity and crystallization.


    Acknowledgments
 
We thank the EMBL team for help in data collection at beamline X11 at the EMBL-Outstation, Hamburg. This project was supported by the Graduiertenkolleg Strukturbildung in makromolekularen Systemen, by the Deutsche Forschungsgemeinschaft and by the Fonds der Chemischen Industrie.


    Notes
 
3 To whom correspondence should be addressed. E-mail: schulz{at}bio5.chemie.uni-freiburg.de Back


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 Introduction
 Materials and methods
 Results and discussion
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Received August 12, 1998; revised October 14, 1998; accepted October 22, 1998.