5
JOURNAL OF BACTERIOLOGY, Nov. 1994, p. 6672-6676 0021-9193/94/$04.00+0 Copyright X 1994, American Society for Microbiology Cloning, Nucleotide Sequence, and Overexpression of the Gene Coding for A5-3-Ketosteroid Isomerase from Pseudomonas putida Biotype B SUHNG WOOK KIM,1 CHAE YOUNG KIM,1 WILLIAM F. BENISEK,2 AND KWAN YONG CHOI`* Department of Life Sciences and Center for Biofunctional Molecules, Pohang University of Science and Technology, Pohang Kyungbuk; Korea,1 and Department of Biological Chemistry, University of Califormia, Davis, Califormia 956162 Received 11 May 1994/Accepted 29 August 1994 The structural gene coding for the A5-3-ketosteroid isomerase (KSI) of Pseudomonas putida biotype B has been cloned, and its entire nucleotide sequence has been determined by a dideoxynucleotide chain termination method. A 2.1-kb DNA fragment containing the ksi gene was cloned from a P. pui biotype B genomic library in Xgtll. The open reading frame of ksi encodes 393 nucleotides, and the amino acid sequence deduced from the nucleotide sequence agrees with the directly determined amino acid sequence (K. Linden and W. F. Benisek, J. Biol. Chem. 261:6454-6460, 1986). A putative purine-rich ribosome binding site was found 8 bp upstream of the ATG start codon. Escherichia coli BL21(DE3) transformed with the pKK-KSI plasmid containing the ksi gene expressed a high level of isomerase activity when induced by isopropyl-f3-D- thiogalactopyranoside. KSI was purified to homogeneity by a simple and rapid procedure utilizing fractional precipitation and an affinity column of deoxycholate-ethylenediamine-agarose as a major chromatographic step. The molecular weight of KSI was 14,535 (calculated, 14,536) as determined by electrospray mass spectrometry. The purified KSI showed a specific activity (39,807 tumol min' mg-) and a Km (60 pM) which are close to those of KSI originally obtained from P. putida biotype B. A5-3-Ketosteroid isomerase (KSI) catalyzes the allylic isomerization of the 5,6 double bond of A5-3-ketosteroids to the 4,5 position by stereospecific intramolecular transfer of a proton (1) (Fig. 1). The enzymatic reaction is present in bacteria which can live on steroids as a sole carbon source. Two isomerases from Comamonas testosteroni and Pseudomonas putida biotype B have been the object of continuous investiga- tion for a number of years (2, 16). Much attention has been drawn to KSIs because the reaction involves the abstraction of proton bonded to a carbon atom, a fundamental and elemen- tary process in the mechanism of various enzymatic reactions (5, 20). KSI from P. putida biotype B has been purified and characterized (17-19). The complete primary structure of P. putida biotype B KSI was determined directly by sequencing of the protein (11). The detailed mechanism of C testosteroni KSI has been studied much more intensely than that of P. putida biotype B. KSI of C. testosteroni catalyzes the reaction very efficiently, since its kcatlKn for the most widely employed substrate, 5-androsten-3,17-dione, approaches the diffusion-controlled limit (13). The gene encoding KSI from C. testosteroni has been cloned (3, 8). The sequencing and in vitro site-directed mu- tagenesis of the ksi gene clarified the identity of aspartic acid 38 and its role in the mechanistic action of catalysis. Asp-38 was strongly suggested to be the general base responsible for abstracting the 41 proton in the steroid substrate from studies on the site-directed mutagenesis of Asp-38 either to asparagine (22) or to glutamic acid (24). Another mutagenesis of tyrosine 14 to phenylalanine suggested that Tyr-14 may act as the general acid protonating the 3-carbonyl oxygen of the keto- * Corresponding author. Mailing address: Department of Life Sci- ences, Pohang University of Science and Technology, Hyoja-dong, Pohang, Kyungbuk, Korea. Phone: (82-562) 279-2295. Fax: (82-562) 279-2199. steroid to form a dienolic intermediate during the catalysis (23). Therefore, both Asp-38 and Tyr-14 seem to be important amino acids involved in the enzymatic reaction of KSI. The C. testosteroni KSI structure has been studied by X-ray crystallog- raphy. Three-dimensional structure of the KSI is known at 6-A (0.6-nm; 1 A = 0.1 nm) resolution (21). A preliminary, unrefined model has been obtained on the basis of a 2.5-A resolution electron density map (9). However, this model possesses an unacceptably high R-factor, and efforts to refine this structure in order to lower the R-factor have not been successful (hla). Part of the difficulty in solving the structure of C. testosteroni KSI arises from the very large size and anisot- ropy of the unit cell [P6(1)22; a = b = 65.4 A, c = 504 A], resulting in technical difficulties in obtaining accurate measure- ments of the structure factor amplitudes. Efforts to find crystallization conditions which provide more suitable crystals for wild-type KSI have not met with success as yet. Thus, a search for homologous KSIs which might crystallize in a more favorable unit cell is of considerable interest. The polypeptide of the KSI from P. putida biotype B is 6 residues longer than that of C. testosteroni KSI. As aligned, there are 44 identical matches, giving only 34% identity between two KSIs (11). A computer search of the sequence databases for proteins homologous to P. putida KSI has yielded no similar proteins other than C. testosteroni KSI. The critical active-site residues of C. testosteroni KSI, Asp-38 and Tyr-14, are conserved in the P. putida KSI polypeptide as Asp-40 and Tyr-16, respectively (reference 11 and this work). The P. putida KSI contains three cysteines and two trypto- phans, whereas the C. testosteroni KSI lacks these amino acids. The P. putida KSI showed dependencies of Vm. and Km on pH that differ from those of C. testosteroni KSI (19). An active- site-directed photoinactivation study of the P. putida KSI implicated the modification of the sulfhydryl group in a cysteine, the extent of which correlated with the observed loss 6672 Vol. 21, No. 11

Cloning, Nucleotide Sequence, and Overexpression of the Gene

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Page 1: Cloning, Nucleotide Sequence, and Overexpression of the Gene

JOURNAL OF BACTERIOLOGY, Nov. 1994, p. 6672-66760021-9193/94/$04.00+0Copyright X 1994, American Society for Microbiology

Cloning, Nucleotide Sequence, and Overexpression of theGene Coding for A5-3-Ketosteroid Isomerase from

Pseudomonas putida Biotype BSUHNG WOOK KIM,1 CHAE YOUNG KIM,1 WILLIAM F. BENISEK,2 AND KWAN YONG CHOI`*

Department of Life Sciences and Center for Biofunctional Molecules, Pohang University of Scienceand Technology, Pohang Kyungbuk; Korea,1 and Department of Biological Chemistry,

University of Califormia, Davis, Califormia 956162

Received 11 May 1994/Accepted 29 August 1994

The structural gene coding for the A5-3-ketosteroid isomerase (KSI) of Pseudomonas putida biotype B hasbeen cloned, and its entire nucleotide sequence has been determined by a dideoxynucleotide chain terminationmethod. A 2.1-kb DNA fragment containing the ksi gene was cloned from a P. pui biotype B genomic libraryin Xgtll. The open reading frame of ksi encodes 393 nucleotides, and the amino acid sequence deduced fromthe nucleotide sequence agrees with the directly determined amino acid sequence (K. Linden and W. F.Benisek, J. Biol. Chem. 261:6454-6460, 1986). A putative purine-rich ribosome binding site was found 8 bpupstream of the ATG start codon. Escherichia coli BL21(DE3) transformed with the pKK-KSI plasmidcontaining the ksi gene expressed a high level of isomerase activity when induced by isopropyl-f3-D-thiogalactopyranoside. KSI was purified to homogeneity by a simple and rapid procedure utilizing fractionalprecipitation and an affinity column of deoxycholate-ethylenediamine-agarose as a major chromatographicstep. The molecular weight of KSI was 14,535 (calculated, 14,536) as determined by electrospray mass

spectrometry. The purified KSI showed a specific activity (39,807 tumol min' mg-) and a Km (60 pM) whichare close to those of KSI originally obtained from P. putida biotype B.

A5-3-Ketosteroid isomerase (KSI) catalyzes the allylicisomerization of the 5,6 double bond of A5-3-ketosteroids tothe 4,5 position by stereospecific intramolecular transfer of a

proton (1) (Fig. 1). The enzymatic reaction is present inbacteria which can live on steroids as a sole carbon source. Twoisomerases from Comamonas testosteroni and Pseudomonasputida biotype B have been the object of continuous investiga-tion for a number of years (2, 16). Much attention has beendrawn to KSIs because the reaction involves the abstraction ofproton bonded to a carbon atom, a fundamental and elemen-tary process in the mechanism of various enzymatic reactions(5, 20). KSI from P. putida biotype B has been purified andcharacterized (17-19). The complete primary structure of P.putida biotype B KSI was determined directly by sequencing ofthe protein (11). The detailed mechanism ofC testosteroni KSIhas been studied much more intensely than that of P. putidabiotype B.KSI of C. testosteroni catalyzes the reaction very efficiently,

since its kcatlKn for the most widely employed substrate,5-androsten-3,17-dione, approaches the diffusion-controlledlimit (13). The gene encoding KSI from C. testosteroni has beencloned (3, 8). The sequencing and in vitro site-directed mu-

tagenesis of the ksi gene clarified the identity of aspartic acid38 and its role in the mechanistic action of catalysis. Asp-38was strongly suggested to be the general base responsible forabstracting the 41 proton in the steroid substrate from studieson the site-directed mutagenesis of Asp-38 either to asparagine(22) or to glutamic acid (24). Another mutagenesis of tyrosine14 to phenylalanine suggested that Tyr-14 may act as thegeneral acid protonating the 3-carbonyl oxygen of the keto-

* Corresponding author. Mailing address: Department of Life Sci-ences, Pohang University of Science and Technology, Hyoja-dong,Pohang, Kyungbuk, Korea. Phone: (82-562) 279-2295. Fax: (82-562)279-2199.

steroid to form a dienolic intermediate during the catalysis(23). Therefore, both Asp-38 and Tyr-14 seem to be importantamino acids involved in the enzymatic reaction of KSI. The C.testosteroni KSI structure has been studied by X-ray crystallog-raphy. Three-dimensional structure of the KSI is known at 6-A(0.6-nm; 1 A = 0.1 nm) resolution (21). A preliminary,unrefined model has been obtained on the basis of a 2.5-Aresolution electron density map (9). However, this modelpossesses an unacceptably high R-factor, and efforts to refinethis structure in order to lower the R-factor have not beensuccessful (hla). Part of the difficulty in solving the structure ofC. testosteroni KSI arises from the very large size and anisot-ropy of the unit cell [P6(1)22; a = b = 65.4 A, c = 504 A],resulting in technical difficulties in obtaining accurate measure-ments of the structure factor amplitudes. Efforts to findcrystallization conditions which provide more suitable crystalsfor wild-type KSI have not met with success as yet. Thus, a

search for homologous KSIs which might crystallize in a more

favorable unit cell is of considerable interest.The polypeptide of the KSI from P. putida biotype B is 6

residues longer than that of C. testosteroni KSI. As aligned,there are 44 identical matches, giving only 34% identitybetween two KSIs (11). A computer search of the sequencedatabases for proteins homologous to P. putida KSI has yieldedno similar proteins other than C. testosteroni KSI. The criticalactive-site residues of C. testosteroni KSI, Asp-38 and Tyr-14,are conserved in the P. putida KSI polypeptide as Asp-40 andTyr-16, respectively (reference 11 and this work).The P. putida KSI contains three cysteines and two trypto-

phans, whereas the C. testosteroni KSI lacks these amino acids.The P. putida KSI showed dependencies of Vm. andKm on pHthat differ from those of C. testosteroni KSI (19). An active-site-directed photoinactivation study of the P. putida KSIimplicated the modification of the sulfhydryl group in a

cysteine, the extent of which correlated with the observed loss

6672

Vol. 21, No. 11

Page 2: Cloning, Nucleotide Sequence, and Overexpression of the Gene

A5-3-KETOSTEROID ISOMERASE FROM P. PUTIDA BIOTYPE B 6673

0

KSI

A B

FIG. 1. Reaction catalyzed by KSI from C. testosteroni. The reac-tion is known to be stereospecific; the beta proton at C-4 is transferredto the beta side of C-6 during the isomerization reaction ofA to B. Thesubstrate is androst-5-ene-3,17-dione and the product is androst-4-ene-3,17-dione.

of enzyme activity (17). The position of the photolabeledcysteine in the primary structure of the P. putida KSI has notbeen identified. Further work on P. putida KSI has beenseverely hampered by the fact that the strain of cells fromwhich the enzyme had been purified had become nonviable instorage. Since the strain had not been deposited with theAmerican Type Culture Collection or another similar reposi-tory for cells, the supply of P. putida KSI was cut off.

Consequently, in order to provide a new source of thisisomerase for further study by X-ray crystallography, to clarifythe difference of enzymatic reaction mechanisms between thetwo KSIs, and to evaluate the role, if any, of cysteine in KSIcatalysis by applying the powerful approach of in vitro site-directed mutagenesis, the gene encoding KSI from P. putidabiotype B has been cloned and sequenced. KSI was overpro-duced in Escherichia coli transformed with an expressionplasmid containing the ksi gene, purified to homogeneity, andcharacterized.

MATERIALS AND METHODS

Bacterial strains and plasmids. A strain of P. putida biotypeB (identified by M. P. Starr, University of California, Davis)was originally isolated from a soil sample by one of us (W.F.B.)but had not been deposited at the American Type CultureCollection. Cell cultures had been maintained on agar mediumcontaining 0.05% testosterone plus the necessary salts andtrace elements (18). This strain of cells became nonviable instorage. Nonviable dried cells obtained from a growth in yeastextract-based medium were used as the source for isolating theksi gene. The E. coli strains used in cloning and expressionwere Y1090(r-) (Promega), DH5aF' (GIBCO-BRL Life Tech-nologies), and BL21(DE3) (Novagen).

Materials. All chemical reagents used were of molecularbiology grade and were obtained from Sigma except forrestriction enzymes, mung bean nuclease, T4 DNA poly-merase, T4 DNA ligase, EcoRI linker, dephosphorylated XgtllEcoRI arms and nick translation system (Promega), the mate-rials used for chromatography, DEAE-Sephacel, SephadexG-50, Sepharose CL-6B, and Superose 12 (Pharmacia), theprotein assay kit and bovine serum albumin standard (Pierce),and the 1-kb DNA ladder size marker (GIBCO-BRL LifeTechnologies). Oligonucleotides used as primers were pur-chased from Korea Biotechnology, Inc.

Preparation of a genomic DNA library. Chromosomal DNAof P. putida biotype B was isolated from the nonviable cells asdescribed elsewhere (6). The genomic DNA fragments biggerthan 3 kb were fractionated by gel electrophoresis on 1.3%low-melting-temperature agarose and recovered from the aga-

rose gel by use of DEAE-Sephacel as described elsewhere (14).After treatment with mung bean nuclease and then T4 DNApolymerase, the genomic DNA was ligated with EcoRI linkerby use of T4 DNA ligase and then digested with EcoRI. DNAfragments (0.5 to 7 kb) were separated by 1.3% low-melting-temperature agarose gel electrophoresis as described aboveand ligated into the dephosphorylated EcoRI sites of XgtllDNA arms. The recombinant Xgtl1 DNA was packaged by useof Packagene E. coli extract (Promega) as recommended bythe supplier.

Preparation of specific DNA probe. In order to isolate apartial DNA fragment of the P. putida ksi gene utilizing theamino acid sequence of P. putida KSI reported previously (11),a PCR was carried out with two mixed oligonucleotides asprimers derived from amino acid residues 30 to 36 and 114 to120, respectively: KSI-1 [5'-CA(A,G) ATG TA(C,T) GC(A,C,G,T) GA(C,T) GA(C,T) GC-3'; 20-mer] and KSI-2 [5'-CCA(A,G)TA (A,C,G,T)GC (C,T)TG CAT (A,C,G,T)GT (C,T)TG-3'; 21-mer]. A PCR was performed for 45 cycles ofdenaturation at 94°C (1 min), annealing at 32°C (1.5 min), andelongation at 72°C (2 min) with a temperature cycler (Eri-comp) in a 100-pul reaction mixture containing 4 ,ug of P. putidagenomic DNA, 1 mM each deoxynucleoside triphosphate(dNTP), 2.4 ,ug of each oligonucleotide primer, and 2.5 U ofAmpliTaq DNA polymerase (Perkin-Elmer Cetus). A singleDNA fragment was amplified from the PCR as judged from asingle band on 1% agarose gel electrophoresis. The amplifiedDNA was phosphorylated at the 5' OH by T4 polynucleotidekinase and ligated into the SmaI site of pBluescript SK(-)(Stratagene) to yield pSK-PCR. The PCR-amplified DNAfragment in pSK-PCR, following restriction enzyme digestion,was separated by 1.3% low-melting-temperature agarose gelelectrophoresis, recovered from a single band in the gel, andthen labeled with 32P by using a nick translation system asrecommended by the supplier. The radiolabeled probe wasseparated from the unincorporated dNTPs with SephadexG-50 as described elsewhere (14).

Screening of the P. putida genomic library by plaque hybrid-ization. E. coli Y1090(r-) transfected with the recombinantAgtll library was plated out on NZCYM-agar plates. Afterincubation of the plates at 37°C for 6 h, plaques were blottedon a dry nitrocellulose filter disk (Schleicher & Schuell). Thenitrocellulose filters were put sequentially on the denaturationsolution (1.5 M NaCl, 0.5 M NaOH) for 10 min and on theneutralization solution (1.5 M NaCl, 0.5 M Tris, pH 8.0) for 5min and then washed with 2X SSC (1lx SSC is 0.15 M NaClplus 0.015 M sodium citrate, pH 7.6) for 5 min while beingshaken. After being dried in the air, the filters were baked at42°C for 12 h, prehybridized in a polyethylene bag containingthe hybridization solution (0.5 M NaH2PO4, 1% bovine serumalbumin [BSA]), 1 mM EDTA, 7% sodium dodecyl sulfate[SDS], pH 7.2) at 65°C for 4 h and hybridized in the samesolution mixed with heat-denatured DNA probe (5 X 107 cpm)at 65°C for 20 h. Positive plaques were detected after the filterswere washed in 2x SSC-0.1% SDS solution at 42°C for 10 mintwice and exposed on X-ray film.

Southern blot hybridization. After DNA fragments obtainedfrom restriction enzyme digestion were run on 1% agarose gel,they were blotted onto a piece of nitrocellulose filter mem-brane (Schleicher & Schuell) as recommended by the supplier.The membrane was dried, baked at 80°C for 2 h under vacuum,and prehybridized. DNA fragments on the membrane werehybridized with the 32P-labeled DNA probe. The prehybrid-ization and hybridization were carried out without usingformamide as recommended (14). To remove the mismatchedprobes, the membrane was washed in preheated washing buffer

VOL. 176, 1994

Page 3: Cloning, Nucleotide Sequence, and Overexpression of the Gene

6674 KIM ET AL.

(2x SSC, 0.1% SDS) for 5 min at 450C. The membrane wasexposed to X-ray film.

Nucleotide sequencing. DNA sequences were determined bythe dideoxynucleotide chain termination method of Sanger etal. (15) with either double-stranded (7) or single-strandedDNA as a template and the Sequenase kit (United StatesBiochemical) as recommended by the supplier. The sequenc-ing primers were SK and Reverse primer oligonucleotides(Stratagene) and synthetic oligonucleotides (KSI-1 to KSI-6).The sequences of the synthetic oligonucleotides KSI-3 to KSI-6used as sequencing primers are as follows: KSI-3, 5'-GC CGCGAG CAG ATT GCC GCG TTC-3'; KSI-4, 5'-G CTC ATCAAA GCG CAT CAC ATC-3'; KSI-5, 5'-AGC CAT AACGGC TGC GGG GCG ATG-3'; KSI-6, 5'-CAT CGC CCCGCA GCC GTT ATG GCT-3'. Double-stranded DNA of therecombinant plasmid was isolated by equilibrium centrifuga-tion in a CsCl-ethidium bromide gradient with a VTi 80 rotor(Beckman) at 60,000 rpm for 10 h at 20°C. Single-strandedDNA of the recombinant pSK plasmid containing the fl originof replication from the fl filamentous phage was isolated byrescuing of single-stranded DNA by coinfection with helperphage M13K07 (Stratagene) as recommended by the supplier.

Analytical methods. Protein concentration was determinedby a protein assay kit with BSA as a standard. In order toprecisely determine the molecular weight of KSI by electro-spray mass spectrometry, the purified KSI was dialyzed exten-sively against distilled water which had been deionized, dilutedin a solution containing 50% acetonitrile and 1% acetic acid tothe final concentration of 20 pmolVll, and then analyzed by anelectrospray mass spectrometer (Model Quattro; VG Biotech).The homogeneities of KSI during purification were assessed bySDS-polyacrylamide gel electrophoresis (PAGE) on a slab gelcontaining 10 to 20% acrylamide in the presence of 0.1% SDSas previously described (10) with subsequent staining withCoomassie blue R250 (Sigma). The electrophoretic molecularweight of KSI was estimated by comparison with low-molecu-lar-weight markers (Sigma).

Determination of KSI activity. The activity of KSI wasdetermined by measuring the product of the enzyme as de-scribed previously (19). 5-Androstene-3,17-dione (Steraloids)was used as the substrate, and the conversion of substrate wasmonitored with time by measuringA248 with a UV spectropho-tometer (Model UV-2100; Shimadzu).

Construction of pKK-KSI overexpressing KSI. A 2.1-kbinsert in the positive recombinant Xgtll DNA was ligated intothe EcoRI site of pBluescript SK(-) (Stratagene) to makepSK-IF. Two DNA primers, a 36-mer (5'-GTT CGA GGTGGC TTG AAT TCA TGA ACC TAC CGA CTG-3') com-plementary to the 5' end of the ketosteroid isomerase-codingregion and a 30-mer (5'-GGC CY[ GCC TGA AGC TTCTAC TGC GGC TCG-3') complementary to the 3' end of thegene, were annealed to the cloned wild-type 393-bp gene inpSK-IF. The large primer contains an EcoRI sequence adja-cent to the ATG start codon of the KSI coding region, and thesmaller primer carries a HindIII sequence just after the TAGstop codon of the ksi gene. A PCR was performed for 35 cycleswith these primers and the GeneAmp DNA AmplificationReagent kit (Perkin-Elmer Cetus) as recommended by thesupplier in a temperature cycler (Ericomp) to amplify the genewith simultaneous introduction of the restriction sites. Afterdigestion with EcoRI and HindIII, the PCR product wasligated into pKK223-3 (Pharmacia) which had been cut withthe same enzymes, to construct pKK-KSI.

Purification of KSI. E. coli transformed with pKK-KSI wasgrown in a shaking flask for 13 to 16 h at 37°C in Luria-Bertanimedium containing 75 mg of ampicillin per liter, and the

expression of KSI was induced by addition of 1 mM isopropyl-P-D-thiogalactopyranoside (IPTG) at the start of growth. Cellsobtained from 1 liter of growth medium were sedimented bycentrifugation and disrupted by sonication of the pellet sus-pended in 50 ml of a cold buffer containing 40 mM potassiumphosphate (KPi), 20 mM ,B-mercaptoethanol, and 1 mMEDTA adjusted to a pH value of 7.0. The undissolved debrisfrom disrupted cell extracts was removed by centrifugation.The soluble fraction was mixed with ethanol to make a 50%ethanol solution and incubated on ice for 30 min. Precipitatewas removed by centrifugation, and the supernatant solutionwas then adjusted to 1 mM EDTA, 80% ethanol, and 20 mM3-mercaptoethanol. The solution was allowed to stand at 40C

overnight, during which time a precipitate containing theisomerase formed and settled out. The precipitate of thissolution was collected by centrifugation and extracted with abuffer containing 0.4 M KP1 (pH 7.0), 1 mM EDTA, and 20mM P-mercaptoethanol. The resulting extract was directlyapplied onto an affinity column of deoxycholate-ethylenedi-amine-Sepharose CL-6B equilibrated with 0.4 M KPi, pH 7.0.The affinity column was prepared by coupling deoxycholate toethylenediamine-linked carbonyl diimidazole-activated Sepha-rose CL-6B as described previously (11, 12). The enzyme waseluted with a buffer containing 1 mM KPi (pH 7.0), 25%ethanol, 1 mM EDTA, and 20 mM ,-mercaptoethanol afterthe column was washed with more than 20 column volumes of0.4 M and 1 mM KPi, pH 7.0, sequentially. Fractions contain-ing KSI as determined by assaying for KSI activity were pooled.Contamination by traces of higher-molecular-weight materialwas removed by eluting KSI with 1 mM KPi, pH 7.0, on aSuperose 12 column with a high-performance liquid chroma-tography system (Model System Prep; Pharmacia).

Nucleotide sequence accession number. The nucleotide se-quence data described in this paper have been deposited in theGenBank database under the accession number L13127.

RESULTS AND DISCUSSION

Cloning of the ksi gene. A single DNA fragment wasamplified from the PCR reaction as judged from a single bandon 1% agarose gel electrophoresis during the preparation ofthe specific DNA probe. The entire sequence of the amplifiedDNA in pSK-PCR was determined by the dideoxynucleotidechain termination method with double-stranded DNA as atemplate, either SK or Reverse primer oligonucleotide as aprimer, and a Sequenase kit as described. The sequencecontained a 273-bp DNA sequence matching the codons of theamino acid sequence in the corresponding region of P. putidaKSI as expected. Three different positive plaques from thegenomic DNA library were found when approximately 20,000plaques were screened with the PCR-amplified DNA probe.The insert sizes of three positive Xgtll recombinant DNAfragments were determined to be 2.1 kb, 1.7 kb, and 0.6 kb by1% agarose gel electrophoresis. These DNA inserts werefound to bind to the DNA probe specifically when Southernblot hybridization of the gel described above was performed.

Restriction enzyme mapping. P. putida genomic DNA in-serts from the three positive recombinant Xgtll DNAs weresubcloned into the EcoRI site of pBluescript SK(-). We foundtwo recombinant pBluescript SK(-) plasmids containing thesame 2.1-kb insert with different orientations. When these tworecombinant plasmids, pSK-IF and pSK-IR, were digested withEcoRI, Sall, SmaI, SacI, BamHI, Clal, and EcoRV, the sizes ofDNA fragments generated by pSK-IF were different fromthose generated by pSK-IR. The sizes of DNA fragmentsobtained by digestion of the two recombinant plasmids with

J. BACTERIOL.

Page 4: Cloning, Nucleotide Sequence, and Overexpression of the Gene

O A5-3-KETOSTEROID ISOMERASE FROM P. PUTIDA BIOTYPE B 6675

L M S V B RI I I _/I-

0.2kb

R BC V S M L RII I \I . I I I

FIG. 2. Restriction enzyme maps of the 2.1-kb inserts containingthe ksi gene in pSK-IF (I) and pSK-IR (II). pSK-IF and pSK-IR were

digested with the restriction enzymes as indicated in the maps, and thesizes of the DNA fragments generated were analyzed by 1% agarosegel electrophoresis with a 1-kb DNA ladder size marker as molecularsize standards. The ksi gene was located by Southern blot analysis ofDNA fragments generated by digestion of both recombinant plasmidswith restriction enzymes. The arrow indicates the location of the ksigenes with the directions of the open reading frames. B, BamHI; C,ClaI; L, SalI; M, SmaI; R, EcoRI; S, SacI; V, EcoRV.

those restriction enzymes were consistent with the restrictionenzyme map of the insert as shown in Fig. 2. The orientationsof the inserts in the recombinant plasmids pSK-IF and pSK-IRwith restriction enzyme sites are shown in Fig. 2. The approx-imate location of the ksi gene in the recombinant pBluescriptSK(-) was estimated by Southern blot hybridization of DNAfragments generated by digestion of the recombinant plasmidpSK-IR with various restriction enzymes including SMaI, Sacl,BamHI, EcoRI, Sail, and ClaL. The recombinant pBluescriptplasmids containing either the 1.7-kb (pSK-1.7) or the 0.6-kb(pSK-0.6) insert DNA fragment did not have both ClaI andBamHI sites in the inserts. Therefore, those recombinantplasmids seemed to contain partial sequences of the ksi gene.The extract of E. coli DH5aF' harboring pSK-IF cultured up tostationary phase showed very low KSI enzyme activity (80U/ml), while either E. coli DH5otF' or E. coli DH5aF' harbor-ing the other recombinant plasmid, pSK-IR, with differentorientation of the 2.1-kb insert from that in pSK-IF did notcontain detectable levels of KSI activity when the enzyme assaywas performed.

Determination of the nucleotide sequences of the ksi gene.The ksi genes in both pSK-IF and pSK-IR plasmids weresubjected to extensive sequencing in order to determine thenucleotide sequences on both DNA strands of the ksi gene.The sequences of four synthetic oligonucleotides (KSI-3 toKSI-6) used as sequencing primers were obtained from nucle-otide sequences of the partial ksi gene in pSK-PCR after beingconfirmed by the sequencing of the 1.1-kb SacI fragment ofpSK-IR. An open reading frame of 393 bp starting with ATGat the 5' end and ending with TAG was found to encode theamino acid sequence of KSI reported previously (11), as shownin Fig. 3. The deduced amino acid sequence encoded by the ksigene is shown below the nucleotide sequence. The polypeptidesequence agreed exactly with the directly determined (11)amino acid sequence. A purine-rich sequence 8 bp upstream ofthe 5' ATG is a putative ribosome binding site of the ksi gene.As aligned, the nucleotide sequence of the P. putida ksi geneshowed 50.9% identical matches with that of the C. testosteroniksi gene (4, 8).

Overexpression, purification, and characterization of KSI.The recombinant plasmid pKK-KSI (5.0 kb) carries a tacpromoter, inducible by IPTG, and a ribosome binding site 10bp upstream of the ATG start codon of the isomerase gene.The identity and fidelity of the insert were confirmed bydetermination of nucleotide sequences of the amplified region

ATGAACCTACCGACTGCGCAGGAAGTCCAGGGCCTGATGGCCCGTTACATCGAGCTGGTC 60M N L P T A Q E V Q G L M A R Y I E L V 20

GATGTCGGGGATATCGAGGCGATICGTGCAGATGTACGCCGATGACGCCACGGTICGAAGAC 120D V G D I E A I V Q M Y A D D A T V E D 40

CCGTTTGGCCAGCCGCCGATCCACGGCCGCGAGCAGATTGCCGCGTTCTATCGCCAGGGT 180P F G Q P P I H G R E Q I A A F Y R Q G 60

TTGGGCGGGGGCAAGGTCCGCGCCTGCCTGACCGGGCCGGTACGGGCCAGCCATAACGGC 240L G G G K V R A C L T G P V R A S H N G 80

TGCGGGGCGATGCCGTTTCGCGTCGAGATGGTCTGGAACGGCCAGCCCTGTGCACTGGAT 300C G A M P F R V E M V W N G Q P C A L D 100

GTCATCGATGTGATGCGCTTTGATGAGCACGGCCGGATCCAGACGATGCAAGCCTACTGG 360V I D V M R F D E H G R I Q T M Q A Y W 120

AGCGAGGTICAACCTCAGCGIGCGCGAGCCGCAGTAGGGCTCAGGCAAGGCC 411S E V N L S V R E P Q ... 131

FIG. 3. Nucleotide sequence of the entire ksi gene and its flankingregions. The underlined purine-rich sequence is the putative ribosomebinding site. Asterisks indicate the stop codon. The amino acidsequences derived from the nucleotide sequences are given belowthem. The nucleotides are numbered from base 1 of the initiationcodon, ATG. The amino acids of the ksi gene product are numberedfrom the first amino acid residue.

in pKK-KSI. E. coli BL21(DE3) transformed with pKK-KSIexpressed a very high level of KSI when induced by 1 mMIPTG. The specific KSI activity of the crude lysate afterremoval of undissolved debris from disrupted cells was 4,807U/mg, which is over 170-fold higher expression than theproduction of KSI from fully induced P. putida biotype B withprogesterone (18). E. coli/pKK-KSI contained a major proteinband migrating on SDS-PAGE a little faster than the 14.4-kDamolecular mass marker. E. coli transformed with pKK223-3exhibited no detectable KSI activity.

Since KSI was the major protein in the crude lysate, wesimplified the purification procedures by utilizing an affinitychromatography process as the major step. Rather than usingammonium sulfate precipitation as in the purification proce-dure described previously (18), we used ethanol for fraction-ation of KSI in the first purification step. Fractional precipita-tion of KSI with ethanol in the concentration range of 50 to80% increased the specific activity almost twofold while retain-

1 2 3 4 5 6

16.9 1014.4 No

10.6

FIG. 4. Polyacrylamide gel electrophoresis of KSI during the puri-fication procedure. The active fraction from each purification step was

run on a continuous-gradient polyacrylamide gel (10 to 20%) contain-

ing 0.1% SDS and stained with Coomassie blue R250. Lanes: 1 and 6,

molecular weight standards (103); 2, crude lysate; 3, ethanol precipi-tation; 4, affinity chromatography; 5, Superose 12 chromatography.

RI

TICGCTATGTGGT

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6676 KIM ET AL.

TABLE 1. Purification of KSI from E. coli BL21(DE3)/pKK-KSI

Total YedProtein Sp act cation-Procedure amt of Yield concn (U/mg of factor

KSI (U) (% (mg/mi) protein) (fold)r

Crude lysate 515,200 100 5.34 4,824 1Ethanol precipitation 267,540 51.9 1.48 9,039 1.9Affinity chromatography 212,892 41.3 1.54 34,560 7.2Superose 12 chromatog- 115,920 22.5 2.08 39,807 8.3

raphy

ing the enzyme activity with the KSI content of over 50% ofoverall yield. A major improvement in purification was madewhen an affinity chromatography of deoxycholate-ethylenedia-mine-agarose was carried out. Almost all of the contaminatingproteins were removed in this step, with a little loss (20%) ofthe enzyme as judged from SDS-PAGE analysis. Overall,7.2-fold purification was achieved, as determined by specificactivity measurement, and the KSI protein band was seen asthe most obvious one even if the sample was overloaded, asshown in Fig. 4. The specific activity (34,560 U/mg) in this stepis higher than that (32,251 U/mg) of KSI purified originallyfrom P. putida biotype B (18). Further purification of KSI wascarried out by employment of gel filtration with a Superose 12column. In this final step, the specific activity reached 39,807U/mg with the overall 8.3-fold purification while losing almosthalf of the enzyme, as shown in Table 1.When the purified KSI was characterized by enzyme kinet-

ics, the Km for 5-androstene-3,17-dione as a substrate was 60puM, which is essentially identical to that (59 jxM) obtained forKSI purified from P. putida biotype B (19). Previous attemptsto measure the molecular weight of KSI by SDS-PAGE, gelpermeation, and equilibrium ultracentrifugation showed thatthe range of monomeric molecular weight was 13,000 to 15,500(18). The estimated molecular weight of the purified KSI basedon the SDS-PAGE analysis was in this range. In order todetermine the molecular weight of KSI more accurately, thepurified KSI was analyzed by electrospray mass spectrometry.The molecular weight of the purified KSI was 14,535, whichagrees very well with the molecular weight (14,536) calculatedfrom the amino acid composition of the primary sequence.As a result of the present work, a reliable source for

abundant amounts of P. putida KSI, which may permit a moretractable crystallographic structure study that can be corre-lated with this KSI's functional properties, now exists. Thepresence of three cysteines in the polypeptide affords oppor-tunities for mercury-based heavy-atom isomorphous derivat-ization which are lacking in the cysteineless C. testosteroni KSI.

ACKNOWLEDGMENTS

We thank Jong Hoon Hahn and Yeoun Jin Kim, Center forBiofunctional Molecules, POSTECH, for performing the mass spectralanalyses.

This research was supported by Non Directed Research Fund,Korea Research Foundation (1991), KOSEF(CBM 9201C), a researchgrant for genetic engineering (MOE, Korea, 1993), and NIH grantDK-14729.

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