Proc. Nati. Acad. Sci. USA Vol. 88, pp. 7620-7624, September 1991 Biochemistry

Leukotriene A4 hydrolase: Determination of the three zinc-binding ligands by site-directed mutagenesis and zinc analysis (recombinant leukotriene A4 hydrolase/catalytic zinc site/peptidase activity/polymerase chain reaction mutagenesis)

JUAN F. MEDINA*t, ANDERS WETTERHOLM*, OLOF RADMARK*, ROBERT SHAPIROt, JESPER Z. HAEGGSTR6M*, BERT L. VALLEEO, AND BENGT SAMUELSSON* *Department of Physiological Chemistry, Karolinska Institutet, S-104 01 Stockholm, Sweden; and tCenter for Biochemical and Biophysical Sciences and Medicine, Harvard Medical School, 250 Longwood Avenue, Boston, MA 02115

Contributed by Bengt Samuelsson, May 10, 1991

From the sequence homology to other zinc hydrolases, His-295, His-299, and Glu-318 have been proposed to be the zinc-binding ligands in LTA4 hydrolase (8, 9). To verify this, we have carried out site-directed mutagenesis of each of these three residues and have determined the zinc content and enzyme activities of the mutated proteins.

Three mutants of recombinant mouse leukoABSTRACT triene A4 (LTA4) hydrolase (3.3.2.6) were produced by sitedirected mutagenesis on cDNA. The codons corresponding to His-295, His-299, or Glu-318 were replaced by codons encoding tyrosine, tyrosine, and glutamine, respectively. The mutated cDNAs were expressed in Escherichia coil, and the three mutated proteins were purified to apparent homogeneity. None of these mutants contained sigcant amounts of zinc, as determined by atomic absorption spectrometry, and all of them were practically devoid of both LTA4 hydrolase and peptidase enzyme activities. Nevertheless, the mutated proteins could be positively identified by their immunoreactivities with an antiserum for human LTA4 hydrolase in immunoblot analysis. Site-directed mutagenesis was also carried out on human LTA4 hydrolase cDNA. Codons encoding His-295, His-299, and Glu-318 were replaced by ones encoding tyrosine, leucine, and alanine, respectively, and the three mutants were expressed in E. coi. The LTA4 hydrolase activities of the total soluble proteins produced in these expressions were less than 10% of that obtained for bacteria harboring noumutated cDNA. In agreement with earlier predictions, our experimental data demonstrate that His295, His-299, and Glu-318 constitute the three ligands of the intrinsic zinc atom in LTA4 hydrolase. Additionally, the combined loss of enzyme activities and zinc content in the purified mutated mouse proteins, emphasi the critical role of the zinc atom for catalysis, whereas the virtually identical chromatographic behaviors of the mutated and nonmutated mouse LTA4 hydrolase proteins suggest that the metal is of limited importance for the maintenance of the enzyme tertiary struture.

MATERIALS AND METHODS Materials. T7 sequencing kit, cDNA spun columns (Sephacryl S-300), restriction endonucleases, and T4 DNA ligase were purchased from Pharmacia. Vent DNA polymerase was from New England Biolabs, and an oligonucleotide-directed mutagenesis kit was from Amersham. SeaPlaque (lowgelling-temperature agarose) was purchased from FMC, and Qiagen tips were from Diagen (Dusseldorf, F.R.G.). Oligonucleotides were synthesized by Scandinavian Gene Synthesis (Koping, Sweden). s of Mouse Polymerase Chain Reaction (PCR) Mut LTA4 Hydrolase cDNA. Site-directed mutagenesis on the recombinant pULTA4, constructed for expression of mouse LTA4 hydrolase cDNA in E. coli (6),§ was carried out by PCR mutagenesis (13-16, 18-21); the strategy is outlined in Fig. 1. Two contiguous regions of the cDNA were amplified in two separate PCR reactions by using four different synthetic oligonucleotides as primers (Table 1). Primers A and B were used to obtain the upstream PCR figment; and primers C and D, for the downstream PCR fragment. PCR amplification (22) and extraction/precipitation of PCR fragments were performed as described (6) with minor modifications. Vent DNA polymerase (1 unit/jul; 1 Al) was the enzyme used for the amplification (26 cycles). After phenol/chloroform (1:1, vol/vol) extraction (twice) and ammonium acetate precipitation, the PCR fragments were digested with eitherBstElI or Sph I (Fig. 1). Further extractions with phenol/chloroform (twice) were followed by gel filtrations on Sephacryl S-300 spun columns. The plasmid pULTA4 was digested with both BstEII and Sph I, and the original BstEII/Sph I cDNA fragment was removed by low-gelling-temperature agarose gel electrophoresis, followed by purification with Qiagen tips (6). In the following ligation, the cohesive ends of the appropriate pULTA4 fragment were ligated to the corresponding ends of the upstream and downstream PCR fragments, which in turn became blunt-end-ligated to each other. This blunt-end ligation is probably facilitated by the use of Vent DNA polymerase (from the marine archaeobacterium Thermococcus litoralis, which apparently lacks the 5' -- 3' exonuclease

Leukotriene A4 (LTA4), an unstable intermediate in the biosynthesis of leukotrienes (1), is derived from arachidonic acid via oxidative metabolism catalyzed by 5-lipoxygenase. Hydrolysis of the epoxide moiety of LTA4 by LTA4 hydrolase (EC 3.3.2.6) leads to leukotriene B4 (LTB4), which is an important proinflammatory mediator. LTA4 hydrolase is a cytosolic enzyme originally found in neutrophils. Characteristic features of this enzyme are the "suicide inactivation" occurring during catalysis and the wide tissue distribution (reviewed in refs. 2 and 3). Human and mouse LTA4 hydrolase cDNAs have been cloned (4-6) and expressed in Escherichia coli as active fusion proteins (6, 7). Recently, a zinc-binding motif was identified in the amino acid sequence of human LTA4 hydrolase when it was compared with the corresponding primary structures of certain aminopeptidases and neutral proteases, typified by thermolysin (8, 9). Accordingly, LTA4 hydrolase has been characterized as a zinc metalloenzyme that also possesses peptidase activity (10-12).

Abbreviations: LTA4, leukotriene A4; LTB4, leukotriene B4; PCR, polymerase chain reaction; RT, room temperature. tTo whom reprint requests should be addressed. §The sequence of mouse LTA4 hydrolase cDNA has been deposited in the GenBank data base (accession no. M63848).

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Proc. Natl. Acad. Sci. USA 88 (1991)

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Expression of Recombinant Mouse LTA4 Hydrolase Muwere expressed as described (6) for pULTA4. Thus, E. coli JM101 cells transformed with mutated recombinants were cultured at 37°C in M9 medium (50 mM Na2HPO4/22 mM KH2PO4/9 mM NaCI/20 mM NH4Cl/0.4% glucose) containing 0.2% Casamino acids, 2 mM MgSO4, and 75 ,ug of ampicillin per ml. Isopropyl fi-D-thiogalactopyranoside was added (0.5 mM final concentration) when the absorbance at 600 nm was about 0.2, and the incubation was continued for 2.5 hr. After centrifugation, cells were resuspended in homogenization buffer (50 mM Tris HCl, pH 8/5 mM EDTA/2 mM dithiothreitol/0.5 mM phenylmethylsulfonyl fluoride/60 ,ug of soybean trypsin inhibitor per ml; 1 ml of the buffer per 25 ml of culture) and homogenized by sonication. Finally, the homogenate was centrifuged for 10 min at 10,000 x g. Analysis of LTA4 hydrolase activity and immunoreactive protein. Aliquots (100 ll) of 10,000 x g supernatants were incubated with 40 ,AM LTA4 at RT for 30 sec. Addition of 100 ,l of methanol to stop the reaction and 200 ng of internal standard prostaglandin B1 (PGB1) was followed by solidphase extraction with Chromabond C18 columns (Macherey & Nagel) and further analysis on a Novapack C18 4-,m 5 mm x 100 mm Radial Pak column (Waters) eluted with methanol/ water/trifluoroacetic acid, 72:28:0.007 (vol/vol), at 1.2 ml/ min (6, 26). For assaying LTA4 hydrolase activities of purified mutated proteins, an aliquot of each (2.5 ,ug in 100 ,ul of 10 mM Tris-HCl, pH 8) was incubated with 70 ,uM LTA4 at RT for 15 sec. Enzymatic formation of LTB4 was quantitated from the peak height ratio LTB4/PGB1 as described (27) and included correction for nonenzymatic formation. For immunoblot (Western) analysis, aliquots of 10,000 x g supernatants were electrophoresed on a SDS/10-15% polyacrylamide gradient gel (using a Pharmacia Phast system), blotted onto nitrocellulose (Amersham Hybond C), and incubated with a polyclonal antiserum against human LTA4 hydrolase (28) as described (29). Protein purifications and peptidase assay. Purification of mutated recombinant mouse proteins (H295Y, H299Y, and E318Q) was performed as will be described elsewhere. The purification procedure included ammonium sulfate precipitation, anion-exchange and hydrophobic-interaction chromatographies, and chromatofocusing. Peptidase activity was measured at RT with the substrate alanine 4-nitroanilide (1 mM) (see ref. 11). The buffer, 50 mM Tris-HCI (pH 7.5), was supplemented with 500 mM NaCI because chloride ions have been found to greatly stimulate the peptidase activity (A.W. and J.Z.H., unpublished data). Zinc content of mutated proteins. Zinc determinations were performed by electrothermal atomic absorption spectrometry with a Perkin-Elmer model 5000 atomic absorption tants. Mutated mouse cDNAs

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Leukotriene A4 hydrolase: determination of the three zinc-binding ligands by site-directed mutagenesis and zinc analysis.

Three mutants of recombinant mouse leukotriene A4 (LTA4) hydrolase (3.3.2.6) were produced by site-directed mutagenesis on cDNA. The codons correspond...
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