Identification of amino acid residues essential for catalytic activity of gentisate 1,2-dioxygenase from Pseudomonas alcaligenes NCIB 9867

FEMS Microbiology Letters 204 (2001) 141^146

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Identi¢cation of amino acid residues essential for catalytic activity of gentisate 1,2-dioxygenase from Pseudomonas alcaligenes NCIB 9867 Chi Hung Chua a , Yongmei Feng a , Chew Chieng Yeo a , Hoon Eng Khoo b , Chit Laa Poh a; * a

Programme in Environmental Microbiology, Department of Microbiology, National University of Singapore, 5 Science Drive 2, 117597 Singapore b Department of Biochemistry, Faculty of Medicine, National University of Singapore, Singapore Received 9 July 2001; received in revised form 8 August 2001 ; accepted 9 August 2001 First published online 27 September 2001

Abstract Gentisate 1,2-dioxygenase (GDO, EC 1.13.11.4) is a ring cleavage enzyme that utilizes gentisate as a substrate yielding maleylpyruvate as the ring fission product. Mutant GDOs were generated by both random mutagenesis and site-directed mutagenesis of the gene cloned from Pseudomonas alcaligenes NCIB 9867. Alignment of known GDO sequences indicated the presence of a conserved central core region. Mutations generated within this central core resulted in the complete loss of enzyme activity whereas mutations in the flanking regions yielded GDOs with enzyme activities that were reduced by up to 78%. Site-directed mutagenesis was also performed on a pair of highly conserved HRH and HXH motifs found within this core region. Conversion of these His residues to Asp resulted in the complete loss of catalytic activity. Mutagenesis within the core region could have affected quaternary structure formation as well as cofactor binding. A mutant enzyme with increased catalytic activities was also characterized. ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Gentisate 1,2-dioxygenase; Mutagenesis ; Catalytic activity

1. Introduction Pseudomonas alcaligenes NCIB 9867 (P25X) has the potential to degrade aromatic compounds such as xylenols and halogenated xylenols as substrates. Degradation of such compounds occurs via the gentisate pathway, one of the three main pathways in aromatic hydrocarbon degradation [1,2]. A critical step in the gentisate pathway is the ¢ssion of the gentisate aromatic ring catalyzed by gentisate 1,2-dioxygenase (GDO, EC 1.13.11.4), resulting in the formation of maleylpyruvate. GDO initiates this reaction by destabilizing the aromatic ring, employing Fe2‡ as a cofactor [3]. The complete open reading frame encoding GDO from P. alcaligenes P25X has recently been cloned and sequenced (accession number AF173167). Whilst other ring cleavage dioxygenases such as the intradiol catechol 1,2-dioxygenase and the extradiol catechol 2,3-dioxygenase have been studied in detail with respect to

* Corresponding author. Tel. : +65 8743674; Fax: +65 7766872. E-mail address : [email protected] (C.L. Poh).

their biochemical and structural properties [4^9], gentisate dioxygenases are relatively under-characterized, and the three-dimensional (3D) crystal structure of the enzyme remains to be elucidated. The 3D crystal structures of other forms of aromatic hydrocarbon dioxygenases such as catechol 1,2-dioxygenase and 2,3-dihydroxybiphenyl 1,2-dioxygenase have recently been reported [10,11]. Since such detailed information is not yet available for GDO, information on amino acid residues that are critical for enzyme activity can only be obtained from mutagenesis experiments. Through random mutagenesis, we have developed a mutant GDO enzyme that possesses both enhanced speci¢c activity and substrate a¤nity for gentisate. 2. Materials and methods 2.1. Bacterial strains and plasmids The GDO gene from P. alcaligenes NCIB 9867, designated xlnE, was cloned into either pGEM-T Easy (Prom-

0378-1097 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 1 ) 0 0 3 9 3 - 7

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ega, USA) or pET30a (Novagen, USA) vectors for mutagenesis experiments. Epicurian coli0 XL-1 Red mutator strain (Stratagene, USA) was the strain employed during in vivo mutagenesis experiments. The host strains employed for GDO enzyme assays were Escherichia coli TOP10 and BL21(DE3) pLysS strains for pGEM-T Easy and pET30a constructs, respectively. 2.2. Error-prone PCR mutagenesis of GDO Error-prone PCR ampli¢cation was performed employing the following pair of oligonucleotide primers £anking the wild-type P25X GDO gene: forward primer 5P-GCT CTA GAC AAC GCC CAA AAA GAA-3P and reverse primer 5P-GCT CTA GAT CTT CAT TGA ATC GAT A3P. The oligonucleotides were designed to amplify the entire coding region as well as 123 bases upstream and 93 bases downstream of the coding region. An XbaI restriction enzyme site (underlined) was also included within the primers to allow the insert to be excised for subcloning purposes. PCR ampli¢cation was carried out using a non-proofreading DyNAzyme1 II Taq polymerase (Finnzymes, Finland) with wild-type P25X genomic DNA as the template. The ampli¢ed 1.2-kb fragment was subsequently cloned into the multiple cloning site of the pGEM-T Easy vector and transformed into chemically competent E. coli TOP10 cells. 2.3. XL-1 Red mutagenesis The plasmid construct containing the insert to be mutagenized was transformed into XL-1 Red competent cells. Conventional heat shock protocols were employed and the transformants were subsequently maintained on the selection plates for 1^2 days, depending on the extent of mutation required, as suggested in the manufacturer's instructions. 2.4. Site-directed mutagenesis Site-directed mutagenesis was carried out using the Quickchange1 Site-Directed Mutagenesis kit (Stratagene, USA) as indicated in the manufacturer's instructions. Oligonucleotide primers used in the mutagenesis were designed with 13 bases £anking both sides of the nucleotide to be mutagenized. Mutagenesis of the nucleotide base was veri¢ed by sequencing both strands of the cloned insert. 2.5. DNA sequence analysis Automated DNA sequencing was performed in a PE Applied Biosystems model 377 DNA Sequencer with the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems, USA). The DNA sequence obtained was analyzed using the HIBIO DNA-

SIS program (Hitachi Software Engineering Co., USA). The CLUSTAL W algorithm available at the EMBL website (http://www.ebi.ac.uk) was used for the multiple alignment of sequences. Nucleotide and amino acid sequence homology as well as conserved binding domain searches were executed on the NCBI BLAST server (http:// www.ncbi.nlm.nih.gov/BLAST) with the blastn and blastp algorithms, respectively. 2.6. Enzyme assay and protein determination GDO activity was obtained by measuring spectrophotometrically at 23³C the change in absorbance at 330 nm which corresponds to the formation of maleylpyruvate [12]. The assays were performed in 3-ml reaction volumes of 0.1 M phosphate bu¡er, pH 7.4, containing 0.33 mM gentisic acid. Enzyme extracts were prepared by sonication of bacterial cell pellets in an appropriate volume of sonication bu¡er (10 mM MOPS (pH 7.4) with 10% glycerol). GDO-speci¢c activities were calculated using a molar extinction coe¤cient value of 10.8U103 M31 cm31 . By definition, one enzyme unit is the amount of enzyme required to produce 1 Wmol of maleylpyruvate per min at 23³C. Km values for all the enzymes were calibrated from Lineweaver^Burk plots with a substrate concentration range of 6^ 133 WM. Protein concentrations were determined by the Bradford assay [13] employing bovine serum albumin as a standard. Relative activities of GDO were calculated with the activity of the wild-type GDO gene construct in E. coli TOP10 strains (0.58 U mg31 ) being taken as 100% activity. 3. Results and discussions 3.1. Random mutagenesis of GDO Out of approximately 300 mutants randomly generated via error-prone PCR and XL-1 Red mutagenesis that were screened, 15 individual mutants were identi¢ed for further characterization based upon signi¢cantly altered catalytic activity. A summary of all the mutants characterized is presented in Table 1. From sequencing of the GDO mutants obtained, both single and double point mutations were evident within the xlnE gene. Amino acid substitutions that occurred within the core central region of the enzyme (positions 99^157) led to the total loss (apart from Mut99IT) of detectable enzyme activity (Table 1). In one of the mutants (mutant 157Stop), the loss of activity could be accounted for by the conversion of a lysine residue at position 157 (AAG) to a stop codon (TAG), leading to the possible formation of an inactive truncated polypeptide. In contrast, mutations outside this core region generally produced variant enzymes with reduced speci¢c activities that ranged from 22% to 75% of the wild-type. For exam-

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Table 1 Sequence analysis and relative speci¢c activities of generated mutants Mutant

Amino acid position

Single point mutations Mut1MT 1 Mut3FY 3 Mut16FS 16 Mut41KE 41 Mut99IT 99 Mut112QR 112 Mut126TR 126 Mut128VG 128 Mut157Stop 157 Mut173FL 173 Mut270KE 270 Mut333RQ 333 Mut344EV 344 Double mutations Mut10 73 103 Sp233 3 334 Conserved His residue mutations Mut108HD 108 Mut110HD 110 Mut149HD 149 Mut151HD 151

Amino acid change

Nucleotide change

Relative speci¢c activity (%)a

MetCThr PheCTyr PheCSer LysCGlu lleCThr GlnCArg ThrCArg ValCGly LysCStop PheCLeu LysCGlu ArgCGln GluCVal

ATGCACG TTTCTAT TTCCTCC AAACGAA ATTCACT CAGCCGG ACGCAGG GTCCGGC AAGCTAG TTCCTTA AAACGAA CGGCCAG GAGCGTG

34 22 1.3 50 40 N.D. N.D. N.D. N.D. 1.1 29 75 2.0

ArgCHis GluCAsp PheCLeu ValCAla

CGCCCAC GAACGAT TTTCTTA GTACGCA

N.D.

HisCAsp HisCAsp HisCAsp HisCAsp

CATCGAT CATCGAT CACCGAC CATCGAT

207

N.D. N.D. N.D. N.D.

a

The relative activities of the mutant GDO enzymes were calculated in comparison with the wild-type P25X enzyme which exhibited a speci¢c activity of 0.58 Wmol of product formed min31 mg31 and which was taken as 100%. Mutants with non-detectable GDO activities are designated N.D.

ple, Mut333RQ exhibited a 25% reduction of relative activity, whereas Mut3FY exhibited a 78% reduction in GDO activity. However, three other mutants (Mut16FS, Mut173FL and Mut344EV) with alterations located outside the core region had very low (1.1^2%) but still detectable levels of GDO activity. A double-stranded L-helix domain was found within the central conserved core region of GDO. The role of helical domains as a subunit linker has been reported for catechol 1,2-dioxygenase [11]. The helical zipper domain present in catechol 1,2-dioxygenase is believed to demarcate the molecular dimer axis of the enzyme, thus playing an intricate role in the dimerization of the subunits. As P25X GDO is a homotetrameric enzyme, this domain could similarly serve as the region of interaction between the individual subunits of the enzyme, leading to the formation of a functional holoenzyme. Therefore, any mutation occurring within this conserved core region could result in the alteration of the quaternary structure of the enzyme by preventing the interaction of the individual subunits. An altered quaternary structure would result in a change in the active site conformation of the enzyme or reduce the af¢nity of the enzyme for its cofactor, both of which would have accounted for the drastic loss in enzyme activity. From the alignment of eight GDO amino acid sequences that were available in the database (Fig. 1), a highly conserved central core region is evident. The data obtained via mutational analysis of GDO suggested that this conserved core region is important in enzyme activity. This

hypothesis is supported by the disparity in residual enzyme activities between mutations generated within this central core region and those of residues outside the core region. Although a majority of the mutations in amino acid residues £anking this core region had the potential to reduce GDO activity by up to 78%, mutations within this core region were found to completely abolish enzyme activity. From the mutants obtained via XL-1 Red mutagenesis, one particular clone, Sp233, possessed an approximately two-fold increase in speci¢c activity towards gentisate when compared to the wild-type enzyme (Table 2). Nucleotide sequence analysis indicated that the mutant contained two point mutations outside the GDO core region. The two point mutations resulted in a Phe to Leu conversion at position 3 as well as a Val to Ala conversion at position 334. 3.2. Biochemical characterization of the Sp233 double mutant Mutant Sp233 expressed a GDO-speci¢c activity of 1.198 U mg31 compared to 0.581 U mg31 for the wildtype GDO. The Km value of the mutant enzyme also differed signi¢cantly from that of the P25X wild-type GDO. The enzyme from mutant Sp233 had a Km value of 19.7 WM whereas the wild-type enzyme had a Km value of 121 WM. This represents an approximately six-fold increase in substrate a¤nity for gentisate. Mutant Sp233 also exhibited an apparent Kcat value of 7.81 min31 site31 compared

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Fig. 1. Multiple sequence alignment of predicted GDO amino acid sequences carried out using CLUSTAL W. Aligned sequences are P. alcaligenes strain P25X (accession number AF173167), Pseudomonas aeruginosa PAO1 (accession number AE004674), Sphingomonas sp. RW5 (accession number AJ224977), E. coli O157:H7 (accession number. AE005174), Bacillus halodurans (two copies, designated copy I and II; accession number AP001514), Ralstonia sp. U2 (accession number AP001514), and Haloferax sp. D1227 (accession number AF069949). Amino acid sequences conserved in all seven GDO species are shaded in black, whereas semi-conserved amino acids are shaded gray. The position of conserved double-stranded L-helix domain is as indicated in the ¢gure. Conserved histidine HRH and HXH domains are marked with asterisks.

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Table 2 Biochemical and kinetic properties of wild-type and mutant Sp233 gentisate dioxygenases Parameter

Strain P25X

Sp233

Subunit (Mr ) pH stability range Temperature stability range (³C) Km (WM)a Speci¢c activity (U mg31 )a Kcat (min31 site31 )a Kcat /Km (min31 WM31 )a

39 000 5.0^7.5 Up to 30 121 þ 4 0.58 þ 0.04 7.81 þ 0.50 (6.45 þ 0.65)U1032

39 000 5.0^7.5 Up to 30 19.70 þ 2 1.20 þ 0.07 16.39 þ 0.95 (83.20 þ 13.41)U1032

a

Values indicated are means þ S.E.M. obtained from triplicate measurements. All values were determined at atmospheric O2 saturation. One enzyme unit is de¢ned as the amount of enzyme that produces 1 Wmol of maleylpyruvate per min at 23³C.

to 16.39 min31 site31 for the wild-type enzyme. The kinetic turnover rate, represented by the Kcat value, suggests that the mutant Sp233 enzyme utilizes gentisate at approximately twice the rate of the wild-type enzyme. An apparent Kcat /Km ratio of 83.20U1032 min31 WM31 for mutant Sp233 was obtained, representing an approximately 13fold increase in catalytic e¤ciency when compared with the wild-type enzyme (6.45U1032 min31 WM31 ). A summary of the biochemical properties of the Sp233 mutant as well as the wild-type enzyme is presented in Table 2. Temperature and pH stability assays indicated that the mutant did not di¡er signi¢cantly from the values obtained for the wild-type enzyme. Both enzymes retained approximately 1.2% of their activities after 10 min incubation at 50³C and were stable within a pH range of 5.0^ 7.5. Catalytic enzymes with increased e¤ciencies or with altered substrate speci¢cities have been generated using random mutagenesis [14,15]. Enzymes with broader substrate speci¢cities or enhanced levels of production are important in industrial applications of bioremediation should they be employed in the removal of aromatic by-products. Our results indicate that improved GDOs can be obtained by random mutagenesis. We found XL-1 Red mutagenesis to be particularly well suited for this process as it allows the level of mutation to be adjusted based upon the length of incubation on the selection media. Inserts with two or more point mutations can be obtained by prolonged incubation of the transformed XL-1 Red strains. 3.3. Conserved His residue mutations Multiple amino acid sequence alignment of the P25Xencoded GDO with other known GDO sequences in the database (Fig. 1) also showed the presence of four highly conserved His residues at positions 108, 110, 149 and 151. Histidine residues have been shown to play a direct role in Fe2‡ coordination in both biphenyl dioxygenase and catechol 1,2-dioxygenase [10,11]. These highly conserved His residues may thus serve a similar function in GDO and were individually targeted for site-directed mutagenesis. The mutation of each of these individual basic His resi-

dues to an acidic Asp residue resulted in the loss of GDO activity to non-detectable levels. The importance of ferrous iron in the reduced state (Fe2‡ ) as a cofactor in GDO activity has been reported [3,16]. Histidine residues of several dioxygenases known to be vital in iron coordination include His residues 153 and 214 in catechol 2,3-dioxygenase [17] and His 145, His 194 and His 209 in 2,3-dihydroxybiphenyl 1,2-dioxygenase [10]. It is therefore reasonable to postulate that the highly conserved His residues in the HRH and HXH motifs within the GDO central core region may play a similar role in Fe2‡ binding. Site-directed mutagenesis carried out to convert these His residues to Asp did indeed result in the complete loss of detectable GDO activity. Although this result clearly indicates that the conserved His residues are integral to the catalytic activity of GDO, additional insight into the actual role of these residues in catalysis would require data from electron paramagnetic resonance spectroscopy as well as X-ray crystallography. 3.4. Conclusion In conclusion, the mutagenesis of P25X GDO indicated that the highly conserved central core region of the enzyme plays an integral role in enzyme activity. This core region could possibly function in the formation of a functional holoenzyme and could also be involved in cofactor binding. The results obtained from this study could thus serve as an approximate guide in the mutagenic studies of other GDOs until the 3D structure of GDO is resolved. Acknowledgements This work was supported by National University of Singapore Academic Research Grant R-182-000-041-112 awarded to C.L.P.

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