- Email: [email protected]
Glutamine-230 influences enzyme solubility but not catalysis in Streptomyces clavuligerus isopenicillin N synthase
FEMS Microbiology Letters 173 (1999) 439^443
Glutamine-230 in£uences enzyme solubility but not catalysis in Streptomyces clavuligerus isopenicillin N synthase Paxton Loke, Tiow-Suan Sim * Department of Microbiology, Faculty of Medicine, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260, Singapore Received 12 February 1999 ; accepted 19 February 1999
Abstract The conversion of N-(L-K-aminoadipyl)-L-cysteinyl-D-valine to isopenicillin N is dependent upon the catalytic action of isopenicillin N synthase (IPNS), an important enzyme in the penicillin and cephalosporin biosynthetic pathway. Recent catalytic investigations on the conserved glutamine-230 in the bacterial Streptomyces jumonjinensis IPNS and the corresponding glutamine-234 in the fungal Cephalosporium acremonium IPNS showed contrasting results whereby the former was suggested to be essential for IPNS activity whereas the latter was found not to be so. In order to unravel these conflicting results, we report the site-directed mutagenesis investigation on the corresponding glutamine-230 in a third IPNS isozyme, which is the bacterial Streptomyces clavuligerus IPNS (scIPNS). IPNS enzymatic assays showed that catalytic activity of the mutant Q230L scIPNS was reduced but not eliminated. Moreover, the solubility of the mutant enzyme was also markedly reduced. Hence, we can conclude that glutamine-230 in scIPNS is not essential for catalysis and correspondingly in all IPNS. z 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Isopenicillin N synthase; Site-directed mutagenesis; Glutamine ; Streptomyces clavuligerus
1. Introduction Streptomyces clavuligerus is an important producer of L-lactam antibiotics such as penicillin N, cephamycin C and clavulanic acid [1]. As such, extensive cloning and heterologous expression of genes involved in the penicillin and cephalosporin antibiotic biosynthetic pathway have been performed in order to understand the biosynthesis of these important compounds [2]. Isopenicillin N synthase (IPNS) is
* Corresponding author. Tel.: 65-8743280; Fax: 65-7766872; E-mail: [email protected]
one of the key enzymes in the penicillin and cephalosporin biosynthetic pathway which catalyzes the formation of isopenicillin N (IPN) from the acyclic peptide N-(L-K-aminoadipyl)-L-cysteinyl-D-valine (ACV) (see Fig. 1) [3]. IPN has both the L-lactam and thiazolidine rings and is the ¢rst L-lactam intermediate formed in the pathway. The initial Aspergillus nidulans IPNS crystal structure proposed that four amino acids, namely, histidine-214, aspartate-216, histidine-270 and glutamine330 are essential for IPNS catalysis [4]. The two histidine and one aspartate residues have since been ¢rmly established as catalytic ligands essential for IPNS activity [5^7]. However, the penultimate glutamine-330 in A. nidulans IPNS and the corresponding
0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 1 0 2 - 0
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P. Loke, T.-S. Sim / FEMS Microbiology Letters 173 (1999) 439^443
glutamines in Streptomyces jumonjinensis IPNS (glutamine-328) and Cephalosporium acremonium IPNS (glutamine-337) respectively were shown by separate groups including ours to be not essential for catalytic activity [8^10]. Thus, it is almost unequivocal that this particular glutamine does not play a crucial role in IPNS catalysis. Further investigations into the role of conserved glutamines in IPNS catalysis again revealed contrasting results for another glutamine residue. The residue glutamine-230 was ¢rst proposed to be essential for IPNS catalysis in S. jumonjinensis IPNS (sjIPNS) [9] but our recent results on the corresponding glutamine-234 in C. acremonium IPNS (cIPNS) showed retention of catalytic activity, implying that this residue is not crucial for activity [11]. Hence, this study attempts to resolve the predicament by choosing a third IPNS species as the arbitrator. The corresponding glutamine-230 in S. clavuligerus IPNS (scIPNS) was altered and the mutant Q230L scIPNS exhibited a reduction of approximately 86% in catalytic activity (based on IPNS protein expressed). Thus, glutamine-230 is not essential for scIPNS catalysis.
pSS971 carrying the scIPNS gene under the control of the T7 promoter (B.J. Sim and T.S. Sim, unpublished results). Polymerization cycles for mutagenesis were increased up to 25 for our purpose. The entire mutant scIPNS gene was sequenced using the ABI PRISM BigDye terminator cycle sequencing kit (PE Applied Biosystems) according to the manufacturer's instructions to verify that only the speci¢c Q230L mutation was altered. Analysis of the sequencing products was performed by an ABI PRISM 377 DNA sequencer at the National University Medical Institutes of the National University of Singapore. 2.2. Growth and induction of Escherichia coli cultures for the heterologous expression of scIPNS, enzymatic assays, protein determination and scanning densitometry The above procedures were performed as previously described [12]. Relative amounts of expressed wild-type and mutant Q230L scIPNS protein were determined by scanning densitometry using the BioRad GS-700 Imaging Densitometer. 2.3. Immunoblot analysis
2. Materials and methods 2.1. Site-directed mutagenesis and DNA sequencing The mutagenic primers 5P TG CAG AAC CTC CTG GTG GAG ACG G 3P and 5P C CGT CTC CAC CAG GAG GTT CTG CA 3P (base change in bold and underlined) were used to alter glutamine230 of scIPNS to a leucine. The Quik-change sitedirected mutagenesis kit (Stratagene) was used according to the manufacturer's instructions for sitedirected mutagenesis on the expression vector
Wild-type and mutant scIPNS samples were separated using a 10% sodium dodecyl sulfate-polyacrylamide gel and electrophoretically transferred to a nitrocellulose membrane by electroblotting [13]. Polyclonal antibodies were raised in rabbits against wild-type scIPNS and cIPNS in a similar fashion for probing [5]. Color development was facilitated by using goat anti-rabbit IgG serum conjugated with horseradish peroxidase (Dako) and 4-chloro-1-napthol (Gibco) for the detection of antigen-antibody complexes.
Fig. 1. Conversion of ACV to isopenicillin N by IPNS.
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441
Table 1 Activity of the wild-type and mutant scIPNS enzymes determined by the bioassay method using Micrococcus luteus ATCC 381 as the test organism Enzyme type
Soluble protein concentration (mg ml31 )
Total activity (units)
Speci¢c activity (units per mg total soluble proteins)
Relative speci¢c activity based on total soluble proteins
Relative % of IPNS expressed
Relative speci¢c activity based on IPNS protein expressed
Wild-type scIPNS Q230L mutant
6.62
0.24
0.0363
100%
100%
100%
6.22
0.0076
0.00122
3.36%
24.3%
13.83%
One unit of activity is the amount of IPNS required to form the equivalent of 1 Wmol of isopenicillin N per ml per min at 26³C. Relative speci¢c activity is expressed as a percentage of the speci¢c activity relative to that of the wild-type enzyme. Wild-type scIPNS expressed is taken as 100% of the relative % of IPNS expressed (determined by scanning densitometry).
2.4. Computer analysis of wild-type and mutant scIPNS protein sequences
not shown). This is probably due to the presence of common antigenic epitopes in IPNS proteins.
The wild-type and mutant scIPNS protein were analyzed using secondary structure prediction programs such as PEPTIDE STRUCTURE and PLOT STRUCTURE which are based on the Chou-Fasman [14] and Garnier-Osguthrope-Robson [15] algorithms. These programs are part of the Genetics Computer Group sequence analysis software package version 7.31 (University of Wisconsin) [16] available at the Bioinformatics Centre of the National University of Singapore.
3.2. Determination of wild-type and mutant Q230L scIPNS activity Soluble protein fractions of wild-type and mutant Q230L scIPNS were used for the determination of enzymatic activity. A relative speci¢c activity of 13.83% (based on IPNS protein expressed) was exhibited by the mutant Q230L (Table 1). Repeated experiments were performed and similar results were obtained.
3. Results 3.1. Expression and immunoblot analysis of wild-type and mutant Q230L scIPNS enzymes As shown in Fig. 2, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that soluble expression of the mutant Q230L scIPNS was signi¢cantly reduced as compared to wild-type scIPNS. The relative amounts of soluble wild-type and mutant scIPNS measured by scanning densitometry were 28.4% and 6.91% respectively. Using immunoblot analyses with both scIPNS and cIPNS antibodies, the authenticity of the scIPNS proteins and their presence in the soluble fraction were veri¢ed and we also found that both scIPNS and cIPNS antibodies showed adequate responses in their immunoreactivity with scIPNS proteins (data
Fig. 2. SDS-PAGE analysis of the soluble protein fractions of wild-type and mutant Q230L scIPNS obtained from Escherichia coli BL21(DE3). Lane 1, molecular mass markers (in kDa) ; lane 2, wild-type scIPNS expressed at 25³C for 15 h; lanes 3^4, two identical mutants Q234L scIPNS expressed at 25³C for 15 h. The arrowhead indicates the position of the scIPNS protein.
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P. Loke, T.-S. Sim / FEMS Microbiology Letters 173 (1999) 439^443
3.3. Computational secondary structure analyses of wild-type and mutant scIPNS Secondary structure analyses of the mutant Q230L scIPNS based on the Chou-Fasman and Garnier-Osguthrope-Robson algorithms showed no obvious change in the overall predicted conformation (results not shown). Hence, it is unlikely that the modi¢cation of glutamine-230 will cause any disruption to the protein secondary structure conformation.
4. Discussion S. clavuligerus IPNS (scIPNS) was chosen as the arbitrating IPNS isozyme because of its striking nucleotide and amino acid sequence similarity of 85% and 81% respectively to sjIPNS [17]. This is in contrast to the fungal cIPNS which shares a lower nucleotide and amino acid sequence similarity of 68% and 60% respectively to the bacterial sjIPNS [17]. Furthermore, phylogenetic studies reveal that scIPNS and sjIPNS are clustered together and support the notion that they are evolutionarily closer to each other than to fungal cIPNS [18]. As glutamine-230 is conserved throughout all IPNS isozymes, its role in the structure and function of IPNS is expected to be similar. For example, the HisXAsp(53^57)XHis motif [6] essential for catalysis has also been proven likewise among IPNS isozymes and in other related non-heme iron dioxygenases [19,20]. Similarly, the conserved penultimate glutamine in three IPNS species has also been proven to be non-essential for activity [8^10]. As such, the results presented in this study strongly support the conclusion that glutamine-230 is not essential for IPNS catalysis as opposed to a previous suggestion [9]. Furthermore, the soluble expression of mutant Q230L scIPNS, although reduced, was still su¤cient for the detection of enzymatic activity. Given the low activity retention of the mutant Q230L scIPNS, this observation would probably be similar in the mutant sjIPNS as both isozymes are very closely related. Thus, it is very likely that this low activity was lost after undergoing harsh denaturation and renaturation conditions with strong chaotropic reagents such as 6 M urea or 8 M guanidinium HCl during the resolubilization process.
It was also evident from the expression studies that glutamine-230 is likely to be crucial for the soluble expression of scIPNS. In our recent study, the alteration of the corresponding glutamine-234 in the fungal cIPNS rendered the mutant cIPNS insoluble at the expected wild-type conditions for expression, which is at 37³C [11]. A reduction of expression temperature to 25³C was needed to overcome this obstacle. Likewise, the solubility of mutant Q230L scIPNS was considerably decreased from 28% (wild-type levels) to approximately 7%. This observation is also consistent with the comment that fungal IPNS is generally more soluble than bacterial IPNS [12]. In conclusion, these results emphasize the fact that resolving the role of a conserved residue in contradictory cases would need at least two con¢rmatory investigations in di¡erent enzymes. As seen here, it cannot be overemphasized that evidence of catalytic studies on site-directed mutagenesis in scIPNS and cIPNS, coupled with the fact that soluble protein is preferred in enzymatic determination, ¢rmly revokes the involvement of glutamine-230 in IPNS catalysis. Furthermore, when the enzymatic activity level is very low, obtaining soluble protein for catalytic analysis is critical, so as to avoid potential errors due to the insoluble aggregation of proteins going through the resolubilization and refolding process.
Acknowledgments We thank Maurice Chan for assistance in preparing the scIPNS antibodies. This work was supported by a National University of Singapore Research Grant no. RP950390/N.
References [1] Jensen, S.E. (1986) Biosynthesis of cephalosporins. Crit. Rev. Biotechnol. 3, 277^301. [2] Miller, J.R. and Ingolia, T.D. (1989) Cloning and characterization of beta-lactam biosynthetic genes. Mol. Microbiol. 3 (5), 689^695. [3] Baldwin, J.E. and Abraham, E.P. (1988) The biosynthesis of penicillins and cephalosporins. Nat. Prod. Rep. 5, 129^145. [4] Roach, P.L., Clifton, I.J., Fulop, V., Harlos, K., Barton, G.J., Hadju, J., Anderson, I., Scho¢eld, C.J. and Baldwin, J.E.
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[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
(1995) Crystal structure of isopenicillin N synthase is the ¢rst from a new structural family of enzymes. Nature 375, 700^ 704. Tan, S.H.D. and Sim, T.S. (1996) Functional analysis of conserved histidine residues in Cephalosporium acremonium Isopenicillin N synthase by site-directed mutagenesis. J. Biol. Chem. 271 (2), 889^894. Borovok, I., Landman, O., Kreisberg-Zakarin, R., Aharonowitz, Y. and Cohen, G. (1996) Ferrous active site of Isopenicillin N synthase: Genetic and Sequence Analysis of the endogenous ligands. Biochemistry 35 (6), 1981^1987. Loke, P., Sim, J. and Sim, T.S. (1997) Functional analysis of a conserved aspartate D218 in Cephalosporium acremonium Isopenicillin N synthase. FEMS Microbiol. Lett. 157, 137^ 140. Sami, M., Brown, T.J.N., Roach, P.L., Scho¢eld, C.J. and Baldwin, J.E. (1997) Glutamine-330 is not essential for activity in isopenicillin N synthase from Aspergillus nidulans. FEBS Lett. 405, 191^194. Landman, O., Borovok, I., Aharonowitz, Y. and Cohen, G. (1997) The glutamine ligand in the ferrous iron active site of isopenicillin N synthase of Streptomyces jumonjinensis is not essential for catalysis. FEBS Lett. 405, 172^174. Loke, P. and Sim, T.S. (1998) Analysis of glutamines in catalysis in Cephalosporium acremonium isopenicillin N synthase by site-directed mutagenesis. Biochem. Biophys. Res. Commun. 252, 472^475. Loke, P. and Sim, T.S. (1998) Catalytic activity in Cephalosporium acremonium isopenicillin N synthase does not involve glutamine-234. Biochem. Biophys. Res. Commun. 248, 559^ 561. Sim, B.J., Tan, D.S.H., Liu, X. and Sim, T.S. (1996) Produc-
[13]
[14] [15]
[16]
[17]
[18]
[19]
[20]
443
tion of high levels of soluble recombinant Streptomyces clavuligerus isopenicillin N synthase in Escherichia coli. J. Mol. Catal. B Enzym. 2, 71^83. Towbin, H., Staehelin, T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. Proc. Natl. Acad. Sci. USA 76, 4350^4354. Chou, P.Y. and Fasman, G.D. (1974) Prediction of protein conformation. Biochemistry 13, 222^245. Garnier, J., Osguthorpe, D.J. and Robson, B. (1978) Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. J. Mol. Biol. 120, 97^120. Devereux, J., Haeberli, P. and Smithies, O. (1984) A comprehensive set of sequence analysis programs for VAX. Nucleic Acids Res. 12, 387^395. Shi¡man, D., Mevarech, M., Jensen, S.E., Cohen, G. and Aharonowitz, Y. (1988) Cloning and comparative sequence analysis of the gene coding for isopenicillin N synthase in Streptomyces. Mol. Gen. Genet. 214, 562^569. Cohen, G., Shi¡man, D., Mevarech, M. and Aharonowitz, Y. (1990) Microbial isopenicillin N synthase genes: structure, function, diversity and evolution. Trends Biotechnol. 8, 105^ 111. Lukacin, R. and Britsch, L. (1997) Identi¢cation of strictly conserved histidine and arginine residues as part of the active site in Petunia hybrida £avanone 3L-hydroxylase. Eur. J. Biochem. 249, 748^757. Shaw, J.F., Chou, Y.S., Chang, R.C. and Yang, S.F. (1996) Characterization of the ferrous ion binding sites of apple 1-aminocyclopropane-1-carboxylate oxidase by site-directed mutagenesis. Biochem. Biophys. Res. Commun. 225 (3), 697^700.
FEMSLE 8699 25-3-99
Glutamine-230 in£uences enzyme solubility but not catalysis in Streptomyces clavuligerus isopenicillin N synthase Paxton Loke, Tiow-Suan Sim * Department of Microbiology, Faculty of Medicine, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260, Singapore Received 12 February 1999 ; accepted 19 February 1999
Abstract The conversion of N-(L-K-aminoadipyl)-L-cysteinyl-D-valine to isopenicillin N is dependent upon the catalytic action of isopenicillin N synthase (IPNS), an important enzyme in the penicillin and cephalosporin biosynthetic pathway. Recent catalytic investigations on the conserved glutamine-230 in the bacterial Streptomyces jumonjinensis IPNS and the corresponding glutamine-234 in the fungal Cephalosporium acremonium IPNS showed contrasting results whereby the former was suggested to be essential for IPNS activity whereas the latter was found not to be so. In order to unravel these conflicting results, we report the site-directed mutagenesis investigation on the corresponding glutamine-230 in a third IPNS isozyme, which is the bacterial Streptomyces clavuligerus IPNS (scIPNS). IPNS enzymatic assays showed that catalytic activity of the mutant Q230L scIPNS was reduced but not eliminated. Moreover, the solubility of the mutant enzyme was also markedly reduced. Hence, we can conclude that glutamine-230 in scIPNS is not essential for catalysis and correspondingly in all IPNS. z 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Isopenicillin N synthase; Site-directed mutagenesis; Glutamine ; Streptomyces clavuligerus
1. Introduction Streptomyces clavuligerus is an important producer of L-lactam antibiotics such as penicillin N, cephamycin C and clavulanic acid [1]. As such, extensive cloning and heterologous expression of genes involved in the penicillin and cephalosporin antibiotic biosynthetic pathway have been performed in order to understand the biosynthesis of these important compounds [2]. Isopenicillin N synthase (IPNS) is
* Corresponding author. Tel.: 65-8743280; Fax: 65-7766872; E-mail: [email protected]
one of the key enzymes in the penicillin and cephalosporin biosynthetic pathway which catalyzes the formation of isopenicillin N (IPN) from the acyclic peptide N-(L-K-aminoadipyl)-L-cysteinyl-D-valine (ACV) (see Fig. 1) [3]. IPN has both the L-lactam and thiazolidine rings and is the ¢rst L-lactam intermediate formed in the pathway. The initial Aspergillus nidulans IPNS crystal structure proposed that four amino acids, namely, histidine-214, aspartate-216, histidine-270 and glutamine330 are essential for IPNS catalysis [4]. The two histidine and one aspartate residues have since been ¢rmly established as catalytic ligands essential for IPNS activity [5^7]. However, the penultimate glutamine-330 in A. nidulans IPNS and the corresponding
0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 1 0 2 - 0
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glutamines in Streptomyces jumonjinensis IPNS (glutamine-328) and Cephalosporium acremonium IPNS (glutamine-337) respectively were shown by separate groups including ours to be not essential for catalytic activity [8^10]. Thus, it is almost unequivocal that this particular glutamine does not play a crucial role in IPNS catalysis. Further investigations into the role of conserved glutamines in IPNS catalysis again revealed contrasting results for another glutamine residue. The residue glutamine-230 was ¢rst proposed to be essential for IPNS catalysis in S. jumonjinensis IPNS (sjIPNS) [9] but our recent results on the corresponding glutamine-234 in C. acremonium IPNS (cIPNS) showed retention of catalytic activity, implying that this residue is not crucial for activity [11]. Hence, this study attempts to resolve the predicament by choosing a third IPNS species as the arbitrator. The corresponding glutamine-230 in S. clavuligerus IPNS (scIPNS) was altered and the mutant Q230L scIPNS exhibited a reduction of approximately 86% in catalytic activity (based on IPNS protein expressed). Thus, glutamine-230 is not essential for scIPNS catalysis.
pSS971 carrying the scIPNS gene under the control of the T7 promoter (B.J. Sim and T.S. Sim, unpublished results). Polymerization cycles for mutagenesis were increased up to 25 for our purpose. The entire mutant scIPNS gene was sequenced using the ABI PRISM BigDye terminator cycle sequencing kit (PE Applied Biosystems) according to the manufacturer's instructions to verify that only the speci¢c Q230L mutation was altered. Analysis of the sequencing products was performed by an ABI PRISM 377 DNA sequencer at the National University Medical Institutes of the National University of Singapore. 2.2. Growth and induction of Escherichia coli cultures for the heterologous expression of scIPNS, enzymatic assays, protein determination and scanning densitometry The above procedures were performed as previously described [12]. Relative amounts of expressed wild-type and mutant Q230L scIPNS protein were determined by scanning densitometry using the BioRad GS-700 Imaging Densitometer. 2.3. Immunoblot analysis
2. Materials and methods 2.1. Site-directed mutagenesis and DNA sequencing The mutagenic primers 5P TG CAG AAC CTC CTG GTG GAG ACG G 3P and 5P C CGT CTC CAC CAG GAG GTT CTG CA 3P (base change in bold and underlined) were used to alter glutamine230 of scIPNS to a leucine. The Quik-change sitedirected mutagenesis kit (Stratagene) was used according to the manufacturer's instructions for sitedirected mutagenesis on the expression vector
Wild-type and mutant scIPNS samples were separated using a 10% sodium dodecyl sulfate-polyacrylamide gel and electrophoretically transferred to a nitrocellulose membrane by electroblotting [13]. Polyclonal antibodies were raised in rabbits against wild-type scIPNS and cIPNS in a similar fashion for probing [5]. Color development was facilitated by using goat anti-rabbit IgG serum conjugated with horseradish peroxidase (Dako) and 4-chloro-1-napthol (Gibco) for the detection of antigen-antibody complexes.
Fig. 1. Conversion of ACV to isopenicillin N by IPNS.
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441
Table 1 Activity of the wild-type and mutant scIPNS enzymes determined by the bioassay method using Micrococcus luteus ATCC 381 as the test organism Enzyme type
Soluble protein concentration (mg ml31 )
Total activity (units)
Speci¢c activity (units per mg total soluble proteins)
Relative speci¢c activity based on total soluble proteins
Relative % of IPNS expressed
Relative speci¢c activity based on IPNS protein expressed
Wild-type scIPNS Q230L mutant
6.62
0.24
0.0363
100%
100%
100%
6.22
0.0076
0.00122
3.36%
24.3%
13.83%
One unit of activity is the amount of IPNS required to form the equivalent of 1 Wmol of isopenicillin N per ml per min at 26³C. Relative speci¢c activity is expressed as a percentage of the speci¢c activity relative to that of the wild-type enzyme. Wild-type scIPNS expressed is taken as 100% of the relative % of IPNS expressed (determined by scanning densitometry).
2.4. Computer analysis of wild-type and mutant scIPNS protein sequences
not shown). This is probably due to the presence of common antigenic epitopes in IPNS proteins.
The wild-type and mutant scIPNS protein were analyzed using secondary structure prediction programs such as PEPTIDE STRUCTURE and PLOT STRUCTURE which are based on the Chou-Fasman [14] and Garnier-Osguthrope-Robson [15] algorithms. These programs are part of the Genetics Computer Group sequence analysis software package version 7.31 (University of Wisconsin) [16] available at the Bioinformatics Centre of the National University of Singapore.
3.2. Determination of wild-type and mutant Q230L scIPNS activity Soluble protein fractions of wild-type and mutant Q230L scIPNS were used for the determination of enzymatic activity. A relative speci¢c activity of 13.83% (based on IPNS protein expressed) was exhibited by the mutant Q230L (Table 1). Repeated experiments were performed and similar results were obtained.
3. Results 3.1. Expression and immunoblot analysis of wild-type and mutant Q230L scIPNS enzymes As shown in Fig. 2, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that soluble expression of the mutant Q230L scIPNS was signi¢cantly reduced as compared to wild-type scIPNS. The relative amounts of soluble wild-type and mutant scIPNS measured by scanning densitometry were 28.4% and 6.91% respectively. Using immunoblot analyses with both scIPNS and cIPNS antibodies, the authenticity of the scIPNS proteins and their presence in the soluble fraction were veri¢ed and we also found that both scIPNS and cIPNS antibodies showed adequate responses in their immunoreactivity with scIPNS proteins (data
Fig. 2. SDS-PAGE analysis of the soluble protein fractions of wild-type and mutant Q230L scIPNS obtained from Escherichia coli BL21(DE3). Lane 1, molecular mass markers (in kDa) ; lane 2, wild-type scIPNS expressed at 25³C for 15 h; lanes 3^4, two identical mutants Q234L scIPNS expressed at 25³C for 15 h. The arrowhead indicates the position of the scIPNS protein.
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3.3. Computational secondary structure analyses of wild-type and mutant scIPNS Secondary structure analyses of the mutant Q230L scIPNS based on the Chou-Fasman and Garnier-Osguthrope-Robson algorithms showed no obvious change in the overall predicted conformation (results not shown). Hence, it is unlikely that the modi¢cation of glutamine-230 will cause any disruption to the protein secondary structure conformation.
4. Discussion S. clavuligerus IPNS (scIPNS) was chosen as the arbitrating IPNS isozyme because of its striking nucleotide and amino acid sequence similarity of 85% and 81% respectively to sjIPNS [17]. This is in contrast to the fungal cIPNS which shares a lower nucleotide and amino acid sequence similarity of 68% and 60% respectively to the bacterial sjIPNS [17]. Furthermore, phylogenetic studies reveal that scIPNS and sjIPNS are clustered together and support the notion that they are evolutionarily closer to each other than to fungal cIPNS [18]. As glutamine-230 is conserved throughout all IPNS isozymes, its role in the structure and function of IPNS is expected to be similar. For example, the HisXAsp(53^57)XHis motif [6] essential for catalysis has also been proven likewise among IPNS isozymes and in other related non-heme iron dioxygenases [19,20]. Similarly, the conserved penultimate glutamine in three IPNS species has also been proven to be non-essential for activity [8^10]. As such, the results presented in this study strongly support the conclusion that glutamine-230 is not essential for IPNS catalysis as opposed to a previous suggestion [9]. Furthermore, the soluble expression of mutant Q230L scIPNS, although reduced, was still su¤cient for the detection of enzymatic activity. Given the low activity retention of the mutant Q230L scIPNS, this observation would probably be similar in the mutant sjIPNS as both isozymes are very closely related. Thus, it is very likely that this low activity was lost after undergoing harsh denaturation and renaturation conditions with strong chaotropic reagents such as 6 M urea or 8 M guanidinium HCl during the resolubilization process.
It was also evident from the expression studies that glutamine-230 is likely to be crucial for the soluble expression of scIPNS. In our recent study, the alteration of the corresponding glutamine-234 in the fungal cIPNS rendered the mutant cIPNS insoluble at the expected wild-type conditions for expression, which is at 37³C [11]. A reduction of expression temperature to 25³C was needed to overcome this obstacle. Likewise, the solubility of mutant Q230L scIPNS was considerably decreased from 28% (wild-type levels) to approximately 7%. This observation is also consistent with the comment that fungal IPNS is generally more soluble than bacterial IPNS [12]. In conclusion, these results emphasize the fact that resolving the role of a conserved residue in contradictory cases would need at least two con¢rmatory investigations in di¡erent enzymes. As seen here, it cannot be overemphasized that evidence of catalytic studies on site-directed mutagenesis in scIPNS and cIPNS, coupled with the fact that soluble protein is preferred in enzymatic determination, ¢rmly revokes the involvement of glutamine-230 in IPNS catalysis. Furthermore, when the enzymatic activity level is very low, obtaining soluble protein for catalytic analysis is critical, so as to avoid potential errors due to the insoluble aggregation of proteins going through the resolubilization and refolding process.
Acknowledgments We thank Maurice Chan for assistance in preparing the scIPNS antibodies. This work was supported by a National University of Singapore Research Grant no. RP950390/N.
References [1] Jensen, S.E. (1986) Biosynthesis of cephalosporins. Crit. Rev. Biotechnol. 3, 277^301. [2] Miller, J.R. and Ingolia, T.D. (1989) Cloning and characterization of beta-lactam biosynthetic genes. Mol. Microbiol. 3 (5), 689^695. [3] Baldwin, J.E. and Abraham, E.P. (1988) The biosynthesis of penicillins and cephalosporins. Nat. Prod. Rep. 5, 129^145. [4] Roach, P.L., Clifton, I.J., Fulop, V., Harlos, K., Barton, G.J., Hadju, J., Anderson, I., Scho¢eld, C.J. and Baldwin, J.E.
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[5]
[6]
[7]
[8]
[9]
[10]
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