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Lactone-Ring-Cleaving Enzyme: Genetic Analysis, Novel RNA Editing, and Evolutionary Implications (Hydrolase͞paraoxonase͞superfamily͞evolution)

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Lactone-Ring-Cleaving Enzyme: Genetic Analysis, Novel RNA Editing, and Evolutionary Implications (Hydrolase͞paraoxonase͞superfamily͞evolution) Proc. Natl. Acad. Sci. USA Vol. 95, pp. 12787–12792, October 1998 Applied Biological Sciences Lactone-ring-cleaving enzyme: Genetic analysis, novel RNA editing, and evolutionary implications (hydrolaseyparaoxonaseysuperfamilyyevolution) MICHIHIKO KOBAYASHI*, MAKOTO SHINOHARA,CHIGUSA SAKOH,MICHIHIKO KATAOKA, AND SAKAYU SHIMIZU Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan Communicated by Takayoshi Higuchi, Kyoto University, Kyoto, Japan, September 8, 1998 (received for review May 20, 1998) ABSTRACT A lactonohydrolase from Fusarium oxyspo- from this mixture, e.g., by solvent extraction, the remaining rum AKU 3702 is an enzyme catalyzing the hydrolysis of pantoic acid can be easily converted to D-pantoyl lactone by aldonate lactones to the corresponding aldonic acids. The heating under acidic conditions. Because this enzyme shows amino acid sequences of the NH2 terminus and internal not only high activity but also high stability, it can be called a peptide fragments of the enzyme were determined to prepare ‘‘superbiocatalyst’’. This enzymatic process has been shown to synthetic oligonucleotides as primers for the PCR. An approx- be applicable for the industrial production of D-pantoyl lactone imate 1,000-base genomic DNA fragment thus amplified was (9, 10). used as the probe to clone both genomic DNA and cDNA for In this paper, we report the structures of cDNA and genomic the enzyme. The lactonohydrolase genomic gene consists of six DNA of this lactonohydrolase, a unique type of RNA-editing, exons separated by five short introns. A novel type of RNA its expression in Escherichia coli, and suggest an evolutionary editing, in which lactonohydrolase mRNA included the inser- relationship between a C-O cleaving enzyme and a P-O tion of guanosine and cytidine residues, was observed. The cleaving enzyme. predicted amino acid sequence of the cloned lactonohydrolase cDNA showed significant similarity to those of the glucono- MATERIALS AND METHODS lactonase from Zymomonas mobilis, and paraoxonases from human and rabbit, forming a unique superfamily consisting of Microorganisms and Cultivation. The lactonohydrolase C-O cleaving enzymes and P-O cleaving enzymes. Lactonohy- gene was isolated from F. oxysporum (AKU 3702, Faculty of drolase was expressed under the control of the lac promoter Agriculture, Kyoto University) (8). E. coli JM109 (recA1, in Escherichia coli. endA1, gyrA96, thi-1, hsdR17, supE44, relA1, D(lac-proAB)yF- 9[traD36, proAB, laclqZDM15]), JM105 (endA1, supE, sbcB15, D y 9 1 q D Lactone chemicals such as A-factor (2-isocapryloyl-3R- thi, rpsL, (lac-proAB) F [traD36, proAB , lac I , lacZ M- g 15]), and XL1-Blue MRF9 (D(mcrA)183, D(mcrCB-hsdSMR- hydroxymethyl- -butyrolactone) (1), virginiae butanolide (2), l2 and N-(b-oxo-acyl) homoserine lactone (3–5) play important -mrr)173, endA1, supE44, thi-1, recA, gurA96, relA1, lac, , [F9, proAB, lacIq ZDM15, Tn10,(tetr)]) were the hosts for pUC roles in the biological world. A-factor (6), which is a low y molecular-mass-autoregulating factor, is akin to hormones in plasmid transformation and phage M13 mp18 19 propagation (11). E. coli transformants were grown in 23 YT medium eukaryotes because it switches on several phenotypes (e.g., y y streptomycin production, streptomycin resistance, and sporu- (1.6% tryptone 1% yeast extract 0.5% NaCl) (11). lation) at an extremely low concentration. Similar autoregu- F. oxysporum collected from an agar slant was inoculated lators containing a g-lactone ring in their structures were into a test tube containing 5 ml of a medium consisting of 10 g reported recently (3, 5). Enzymes that synthesize or degrade of glycerol,5gofPolypepton (Daigo, Osaka, Japan),5gof these molecules must tightly control the amounts of such yeast extract (Oriental Yeast, Tokyo, Japan), and5gofcorn lactone autoregulators. However, there have been no reports steep liquor (Shono Starch, Suzuka, Japan) per liter of tap on the structures and functions of lactone-hydrolyzing enzymes water (pH 6.0) and then incubated at 28°C for 24 h with at the molecular level, except for one on the gluconolactonase reciprocal shaking. The subculture was then inoculated into a 2-liter shaking flask containing 500 ml of a medium (pH 6.0) from Zymomonas mobilis (7). y y We previously reported (8) that a fungus, Fusarium oxys- (which consisted of 30 g of glycerol 5 g of yeast extract 5gof porum AKU 3702, produces a unique lactone-hydrolyzing corn steep liquor per liter of tap water), and incubated at 28°C for 4 days with aeration. Cells were harvested by filtration, enzyme (lactonohydrolase) that catalyzes the reversible hy- 2 drolysis of various aldonate lactones and aromatic lactones. washed twice with water, and then stored at 80°C until use. Isolation of Peptide Fragments and Their Sequencing. The purified enzyme stereospecifically hydrolyzes aldonate The lactonohydrolase (30 nmol), which was purified according to lactones such as D-galactono-g-lactone and L-mannono-g- lactone, and also catalyzes the enantioselective hydrolysis of the procedure previously reported (8), was digested with lysylendopeptidase in 50 mM TriszHCl buffer (pH 9.0) con- D-pantoyl lactone, which resembles aldonate lactones in chem- y ical structure. When racemic pantoyl lactone serves as a taining 4 M urea for 12 h at 30°C at a substrate enzyme ratio substrate for the lactonohydrolase of F. oxysporum, only of 100:1. The peptides obtained on this digestion were sepa- rated by high performance liquid chromatography (HPLC) on D-pantoyl lactone can be converted to D-pantoic acid without a Cosmosil 5C -AR column (4.6 by 250 mm; Nacalai Tesque, any modification of the L enantiomer, resulting in the trans- 18 Kyoto, Japan) and eluted with a linear gradient of acetonitrile formation of the racemic mixture into D-pantoic acid and (0–50% by vol) in the presence of 0.1% (by vol) trifluoroacetic L-pantoyl lactone (9). After the removal of L-pantoyl lactone acid at the flow rate of 1.0 mlymin. The peptides isolated were The publication costs of this article were defrayed in part by page charge Data deposition: The sequences reported in this paper have been payment. This article must therefore be hereby marked ‘‘advertisement’’ in deposited in the GenBank, DDBJ, and European Molecular Biology accordance with 18 U.S.C. §1734 solely to indicate this fact. Laboratory (EMBL) databases [accession nos. AB010465 (cDNA) © 1998 by The National Academy of Sciences 0027-8424y98y9512787-6$2.00y0 and AB010980 (genomic DNA)]. PNAS is available online at www.pnas.org. *To whom reprint requests should be addressed. 12787 Downloaded by guest on September 25, 2021 12788 Applied Biological Sciences: Kobayashi et al. Proc. Natl. Acad. Sci. USA 95 (1998) sequenced with a gas-liquid-phase protein sequencer (Applied sequences of the cloned genomic DNA and cDNA for lacto- Biosystems model 477A). nohydrolase were determined in both orientations. Preparation of Genomic DNA. Total DNA of F. oxysporum Determination of 5*-End of the Lactonohydrolase cDNA. was isolated by a modification of the method of Malardier et The nucleotide sequences of the insert DNA in each positive al. (12). The following procedure was carried out: liquid N2 was cDNA clone hybridizing with the probe were determined by added to frozen cells (10–20 g), and the cells were ground with chain termination sequencing (14). However, these cDNA a ceramic pestle and mortar. The ground cells were suspended clones did not contain a start codon, although they contained in 100 ml of 10 mM Hepes (pH 6.9) containing 200 mM EDTA the sequence corresponding to the amino acid sequence and 1% SDS. The mixture was then incubated for2hat65°C. determined from the purified enzyme of F. oxysporum. There- DNA was purified by extracting the lysate with phenoly fore, the rapid amplification of cDNA ends (RACE) method chloroform (1y1; volyvol), precipitated with 2-propanol, was used to extend 59-end of the lactonohydrolase cDNA. The treated with RNase, and reprecipitated with ethanol. cDNA fragment containing the start codon was subcloned by Cloning of a Partial Lactonohydrolase Genomic DNA. Oli- using a Marathon cDNA amplification kit (CLONTECH) and gonucleotide primers were synthesized based on the amino a TA cloning kit (Invitrogen). This fragment was cloned into acid sequences of the NH2 terminus and internal fragments the EcoRI site of pUC18 designated as pFLC40 and se- generated with lysylendopeptidase. The amino acid sequence, quenced. Phe-His-Val-Tyr-Asp-Glu-Glu-Phe-Tyr-Asp, was used to Preparation of Crude Extracts of E. coli Transformants. model the oligodeoxynucleotide pool 59-AAAAGCTTCCA- The recombinant E. coli JM109 containing pFLC40E was C(T)GTCTAC(T)GAC(T)GAA(G)GAA(G)TTC(T)TAC- cultured aerobically to late log phase in 10 ml of 2 3 YT (T)GAC(T)GT-39 (sense strand), and Asp-Gly-Val-His-Val- medium containing 100 mgyml ampicillin in a 100-ml test tube Trp-Asn-Pro to model 59-GGCTTGCTGCAGGGG(A)TTC- at 28°C or 37°C and then transferred to 100 ml of the same CAA(C, G,T)ACG(A)TGA(C, G,T)ACA(C, G,T)CCG(A)T- medium in a 500-ml shaking flask with isopropyl b-thiogalac- C-39 (antisense strand). These oligonucleotides were synthe- topyranoside at a final concentration of 1 mM to induce the lac sized by the phosphoramidite method (13) by using an Applied promoter. After a further 7-h or 12-h cultivation, the cells were Biosystems model 381A automated synthesizer. DNA was harvested by centrifugation, suspended in 4 ml of 0.1 M Pipes amplified by means of the PCR by using a thermal cycler (pH 7.0), disrupted by sonication for 5 min (19 kHz; Insonator (Perkin–Elmer). The reaction mixtures comprised 2.5 mgof model 201M, Kubota, Tokyo), and then centrifuged at template DNA, 250 pmol of each oligonucleotide pool, and 12,000 3 g for 10 min.
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