PEDS Advance Access published online on June 8, 2007
Protein Engineering Design and Selection, doi:10.1093/protein/gzm023
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Concurrent mutations in six amino acids in ß-glucuronidase improve its thermostability
1 Biotechnology Research Institute, Shanghai Academy of Agricultural Sciences, 2901 Beidi Road, Shanghai 201106, China 2 Department of Plant Sciences, University of Tennessee, Knoxville, TN 37996, USA 3 Department of Plant Science, University of Connecticut, Storrs, CT 06269, USA 4 College of Horticulture, Nanjing Agricultural University, 1 Weigang, Nanjing 210095, China 5 College of Bioscience and Biotechnology, Yangzhou University, 88 S. Daxue Road, Yangzhou 225009, China
6 To whom correspondence should be addressed. E-mail: yaoquanhongcn{at}yahoo.com.cn
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Abstract |
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To achieve a thermostable ß-glucuronidase (GUS) and identify key mutation sites, we applied in vitro directed evolution strategy through DNA shuffling and obtained a highly thermostable mutant GUS gene, gus-tr, after four rounds of DNA shuffling and screening. This variant had mutations in 15 nucleic acid sites, resulting in changes in 12 amino acids (AAs). Using gus-tr as the template, we further performed site-directed mutagenesis to reverse the individual mutation to the wild-type protein. We found that six sites (Q493R, T509A, M532T, N550S, G559S and N566S) present in GUS-TR3337, were the key AAs needed to confer its high thermostability. Of these, Q493R and T509A were not reported previously as important residues for thermostability of GUS. Furthermore, all of these six mutations must be present concurrently to confer the high thermostability. We expressed the gus-tr3337 gene and purified the GUS-TR3337 protein that contained the six AA mutations. Compared with the wild-type protein which lost its activity completely after 10 min at 70°C, the mutant GUS-TR3337 protein retained 75% of its activity when heated at 80°C for 10 min. The GUS-TR3337 exhibited high activity even heated at 100°C for 30 min on nitrocellulose filter. The comparison of molecular models of the mutated and wild-type enzyme revealed the relation of protein function and these structural modifications.
Keywords: ß-glucuronidase/directed evolution/enzyme properties/structure-function analysis/thermostability
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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In vitro directed evolution through DNA shuffling is a powerful molecular tool for creation of new biological phenotypes. Stemmer (1994)
first introduced the method of DNA shuffling for in vitro mutation of recombinant genes from a set of parental genes. Since then, the technique has been routinely applied to evolve enzymes for basic research or to improve proteins of industrial significance, such as improved kinetics, altered substrate or product specificities (Zhao et al., 2002; Hibbert et al., 2005; Otten and Quax, 2005), plant improvement (Lassner and Bedbrook, 2001; Dixon et al., 2003; Castle et al., 2004), as well as vaccine and pharmaceutical development (Whalen et al., 2001; Locher et al., 2004, 2005).
ß-glucuronidase (GUS) is the most widely used reporter used in screening for genetically engineered plants, as indicated in the database of 2001 and 2002 of field trials in the USA (Miki et al., 2004). Its popularity is attributed to its high stability in plant tissues and lack of toxicity even when expressed at high levels. Histochemical GUS staining protocol is a simple, rapid, highly reliable and cost-effective method for analysis of transgenic plants (Jefferson et al., 1987). Also, no specialized equipment is needed for histochemical assays of GUS activity. Moreover, the GUS protein is considered safe for humans and the environment (Mantis and Tague, 2000). GUS, therefore, provides an excellent model system for studying in vitro directed evolution and structure-function relationship because of its stability, easily identifiable phenotype (by histochemical staining) and relatively small size. Also, analyses of its enzymatic activity and other biochemical properties can be conveniently performed (Rowe et al., 2003; Xiong et al., 2007).
By directed evolution, Matsumura et al. (1999) isolated a modified GUS protein that was significantly more resistant to both glutaraldehyde and formaldehyde than the wild-type enzyme. This improvement seemed to reduce problems with loss of GUS activity caused by glutaraldehyde or formaldehyde during tissue fixation. Matsumura and Ellington (2001) also used DNA shuffling to direct evolution of E.coli GUS variants with improved ß-galactosidase activity. Through directed evolution, some thermostable GUS mutants have been isolated by DNA shuffling with the best variant retaining activity at 70°C, in comparison to the wild-type enzyme that loses its function at 65°C (Flores and Ellington, 2002; Xiong et al., 2002). A mutant of GUS with increased pNP-xyl activity has also been obtained (Geddie and Matsumura, 2004). Even though the mutated amino acids (AAs) were identified in these studies, none of the mutations were verified for their roles by back conversion of the mutant AA to its wild-type AA.
For this report, we performed in vitro directed evolution and obtained a mutant GUS that was highly thermostable. Using mutation and reversion approaches, we identified six key AAs (Q493R, T509A, M532T, N550S, G559S and N566S) that are responsible for the improved thermostability and provide data for the correlation between their improved thermostabilities and the modified protein structures.
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Materials and methods |
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Gene Shuffling
The details for gene shuffling to generate the thermostable mutant gus-tr were described elsewhere (Xiong et al., 2007). Briefly, the DNA shuffling was performed as described by Stemmer (1994) with modifications as follows: fragments of 50–100 bp were purified from 10% PAGE by electrophoresis using dialysis. After the primerless PCR and primer PCR using the oligonucleotides R3746, R3747 as primers, a collection of full-length gusA mutants was obtained and digested with Bam HI and Sac I enzymes and the isolated fragments were cloned into the pYPX251. The prokaryotic expression vector pYPX251 containing promoter of the aacC1 gene and the T1T2 transcription terminator was constructed in our laboratory and deposited in GenBank (accession number AY178046). The mutant DNA library was delivered into the E.coli strain DH5
by electroporation and plated on LB plates containing 100 µg ml–1 ampicillin. After 16 h of growth at 37°C, colonies were absorbed onto a nitrocellulose filter and transferred colony-side-up to a Petri dish, which were then placed into an oven at temperature 70–110°C for 30 min. The Petri dishes were taken out to ambient temperature (
25°C) for 10 min, and the filter papers were incubated in 0.4 mg ml–1 X-Gluc (5-bromo-4-chloro-3-indolyl-ß-D-glucuronic acid) at 37°C with GUS buffer solution (50 mM sodium phosphate, pH 7.0; 1 mM EDTA; 5 mM ß-mercaptoethanol). The small area containing the blue colonies was cut off using scissors. Each small nitrocellulose filter was placed into a 500 µl tube. After adding 20 µl TE buffer, the tubes were incubated for 10 min at 99°C and then spun briefly. Then, screened and identified gusA variants of a given round were pooled together and used as templates for the generation of further mutants by DNA shuffling.
To determine the role of specific AA mutations in gus-tr in thermostability, site-directed mutagenesis via an overlap extension PCR strategy (Xiong et al., 2004, 2006b; Peng et al., 2006) was used to reverse each of these 12 AA substitutions in GUS-TR to wild-type, and in wild-type to GUS-TR. The strategy for the site-directed mutagenesis was shown in Fig. 1A and B, and the primers used are listed in Supplementary Material, Table S1.
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DNA sequencing
The evolved gusA genes were sequenced using the Applied Biosystems Big Dye protocol with ABI Prism 377 DNA sequencer (Applied Systems Company, Foster City, CA, USA). The 3'-end of every evolved gusA gene was sequenced using the primer W73 on the vector pYPX251. The mutant genes gus-tr and gus-tr3337 were sequenced in their entirety, using the following additional primers: W1062, W1063, W1064, W1065, W1066 (Supplementary Material, Table S1).
Protein purification and quaternary structure determination
Each GUS protein was fused to an N-terminal His6-tag and the GUS-WT, GUS-TR and GUS-TR3337 enzymes were purified to homogeneity using nickel chelate affinity chromatography. Protein preparations were evaluated by SDS–PAGE analysis and Coomassie Blue staining (Sambrook and Russell, 2001). Purified protein concentrations were determined using the Bradford protein assay (Bio-Rad, Herculer, CA). High temperature treatments of purified wild-type and mutant GUSs were carried out using a PE9600 thermocycler (Applied Systems Company, Foster City, CA, USA). The oligomerization state of these GUSs was ascertained by SDS–PAGE according to Flores and Ellington (2002). Samples of GUS protein in the loading buffer (50 mM Tris–HCl (pH 6.8), 0.5% SDS, 10% glycerol, 28 mM ß-mercaptoethanol, 0.05% bromophenol blue) were heated for 5 min at a given temperature. Samples were loaded onto an 8% SDS–PAGE and the proteins were visualized by Coomassie Blue staining.
Enzyme characterization and kinetics
The optimal pH of the enzymes GUS-WT, GUS-TR and GUS-TR3337 were determined at 37°C at every half point between 2.0 and 10.0. The GUS activities were measured at 5° intervals between 20 and 80°C to determine the temperature at which maximum activity was retained. The thermostabilities of the expressed GUSs at 80°C were determined by measuring the activity remaining at 37°C after heating the GUS samples and chilling on ice. Kinetics parameters were determined as described (Matsumura et al., 1999; Flores and Ellington, 2002) with each value derived from at least three independent reactions.
Swiss model was used for molecular modeling of the wild-type E.coli GUS and the GUS-TR3337 site-directed mutants (http://swissmodel.expasy.org/) (Schwede et al., 2003). The three-dimensional structures of wild-type E.coli GUS and site-directed mutant GUS-TR3337 were aligned with the template structure of human GUS whose structure was obtained by X-ray crystallography (Jain et al., 1996).
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Results |
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Directed evolution and screening
Using the DNA shuffling system by Xiong et al. (2007), we shuffled the gusA gene and developed a highly efficiency, high-throughput system for in vitro directed evolution of the gusA reporter gene. Over 500 000 variant colonies were screened using the pYPX251 vector in each round of DNA shuffling and screening. One of the GUS mutants, named GUS-TR, was identified because it was significantly more resistant to high temperature than the wild-type enzyme after four rounds of directed evolution and screening. The GUS-TR protein maintained its activity even when colonies containing it were heated on nitrocellulose filters at 100°C for 30 min. Furthermore, the purified GUS-TR protein retained its activity after heated at 80°C for 30 min.
Characterization of the role of each of altered AA in GUS-TR's thermostability using site-directed mutagenesis
Sequencing of the evolved gus-tr gene showed that the gene had been altered in 15 nucleic acid sites (GenBank accession number DQ513151), which led to 12 AA substitutions: I149T, N181S, D436E, V446A, A451V, Q493R, T509A, M532T, N550S, G559S, N566S and M591I. Based on the strategy shown in Fig. 1A, site-directed mutagenesis reverted the GUS-TR to GUS-WT using the primers list in Supplementary Material, Table S1. As shown in Table 1, the AA residues of Q493R, T509A, M532T, N550S, G559S and N566S were key mutations for thermostability, while I149T, N181S, D436E, V446A, A451V and M591I were not essential because these mutations did not lead to changes in thermostability of the GUS protein (LX16, LX17 and YH1221).
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Using site-directed mutagenesis, we created GUS-TR3337 (GenBank accession number DQ513152) and verified that the mutations in all six sites collectively conferred the thermostability. Individual AA mutations among the six AAs did not yield a significant increase in thermostability (Table 2 and Fig. 1B). The variant of GUS-TR3337, which was mutated at all these six AA residues, was the simplest mutant GUS that conferred thermostability in this study. The GUS-TR and GUS-TR3337 proteins exhibited high activity even when the nitrocellulose filter containing the mutant colonies was heated at 100°C for 30 min (Fig. 2).
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Protein purification and enzyme characterization
Wild-type GUS (GUS-WT) and variants (GUS-TR and GUS-TR3337) were purified to homogeneity using nickel chelate affinity chromatography. With pNP-ß-D-glucuronide (pNPG) as a substrate, the specific activity of GUS-WT, GUS-TR and GUS-TR3337 were approximately 142.9, 51.2 and 43.1 U mg–1 at 37°C and pH 6.5. One single activity peak was found at pH 6.5 in all three GUSs (GUS-WT, GUS-TR, and GUS-TR3337) (Fig. 3A). The optimal temperature for GUS-WT tested at pH 6.5 was 45°C, while that for both GUS-TR and GUS-TR3337 were at 65°C (Fig. 3B). These results demonstrated that the optimal temperatures for the GUS activity of GUS-TR and GUS-TR3337 were increased to 20°C.
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We examined the thermostability of the expressed GUS-WT and observed that the enzyme activity decreased significantly after 10 min at 70°C (Fig. 3C) and 2 min at 80°C (Fig. 3D). However, the expressed GUS-TR retained 88% of its activity when heated at 80°C for 10 min and 56% when heated at 80°C for 30 min. The activity decreased significantly following a heat treatment at 90°C or higher (Fig. 3C). The expressed GUS-TR3337 retained 75% of its activity when heated at 80°C for 10 min and 48% when heated at 80°C for 30 min. Similar to that of GUS-TR, GUS-TR3337's activity decreased significantly following a heat treatment at 90°C or higher (Fig. 3C). GUS-TR3337 was less thermostable than that of GUS-TR (Fig. 3C and D). In general, thermostabilities of enzymes often correlate with their optimal temperatures. Enzymes that show higher thermo resistance often exhibit higher activities at those temperatures (Flores and Ellington, 2002
). GUS-TR and GUS-TR3337 had higher enzymatic activities at 65°C than at 37°C, and were, therefore, more thermo resistant.
We have also shown that wild-type version of GUS (GUS-WT) became monomers under SDS–PAGE with 28 mM ß-mercaptoethanol, 0.5% SDS in loading buffer, 0.1% SDS in gel and 0.1% SDS in electrophoresis buffer. GUS-WT was denatured and became 100% monomeric regardless of the temperature treatments (Fig. 4A). In contrast, the GUS-TR and GUS-TR3337 were quite stable and tetramerized as required for the GUS activity (Jain et al., 1996; Flores and Ellington, 2002). GUS-TR and GUS-TR3337 proteins had much greater portions in quaternary structure at 76°C. GUS-TR and GUS-TR3337 proteins also retained its quaternary structure at 80°C but became monomers at 90°C or higher (Fig. 4B–E). Because monomers of GUS also have much reduced enzymatic activity, it is not surprising that the mutants exhibited some activity at 90°C or higher when the quaternary structure of the enzyme was lost.
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The thermostabilities of GUS-TR and GUS-TR3337 were greatly improved compared to those previously reported (Flores and Ellington, 2002
; Xiong et al., 2002). The detailed kinetic properties of GUS-WT, GUS-TR and GUS-TR3337 were determined with various concentrations of pNPG and simple Michaelis–Menten constants were derived (Table 3). The thermostable GUS-TR and GUS-TR3337 exhibited reductions in Kcat and increases in Km, and hence decreasing the Kcat Km–1 ratios.
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Tubes containing GUS-TR, GUS-TR3337 and GUS-WT proteins in buffer were heated at various temperatures for 10 min and then cooled at ambient temperature (
25°C) for 10 min. The tube containing GUS-WT became cloudy and thick at 50°C but returned to clarity after 10 min at 25°C (Supplementary Material, Fig. S1). At 60°C, the tube containing GUS-WT became cloudy and stayed cloudy even after the tube was placed 10 min at 25°C, indicating that the GUS-WT protein was denatured (Supplementary Material, Fig. S1). The tubes containing GUS-TR and GUS-TR3337 were somewhat cloudy at 80°C, but became clear after 10 min at 25°C (Supplementary Material, Fig. S1). On the other hand, treatment at 80°C for 10 min made the tubes containing the GUS-WT protein stay cloudy and never cleared after being returned to the ambient temperature. This demonstrated that the denaturation of the GUS-WT protein at 80°C was irreversible. All of these data suggest that the GUS-TR and GUS-TR3337 mutant proteins were more thermostable than the GUS-WT protein. Three-dimensional structural analyses of GUS-TR3337
The three-dimensional structures of the mutant GUS GUS-TR3337 and the wild-type GUS were derived using Swiss-Model server, using human GUS as template. Fig. 5A and B showed the predicted positions of the six mutated AA residues (Q493R, T509A, M532T, N550S, G559S and N566S) in the GUS-TR3337 and GUS-WT. N550S is buried, while M532T, G559S and N566S are localized in interface of two monomers. The residue Q493R lies in TIM-barrel domain and near the catalytic residues, and also lies at the tetramer interface. The residue T509A is near the active site in the crystal structure of the homologous human GUS. Comparison of the mutated AA residues sequences and structures of GUS-TR3337 and GUS-WT indicated that Q493R and M532T lie in the alpha helix, while N550S, G559S and N566S lie in the extended strand (Fig. 6).
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: alpha helix; 
: extended strand; 
: turn. |
Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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In vitro directed evolution through DNA shuffling is a powerful molecular tool for creation of new biological phenotypes, such as elevated expression and high thermostability. GUS, one of the most widely used reporter enzymes, provides an excellent model system to study in vitro directed evolution. In this paper, we focused on identifying the key points of a mutant highly thermostable ß-glucuronidase (GUS-TR), which was significantly more resistant to high temperatures than wild-type enzyme. Even at 100°C for 30 min, the colonies exhibited comparative activity when absorbed onto a nitrocellulose filter. The highly thermostable GUS, which displayed twelve AA changes, was isolated after four rounds of DNA shuffling and screening.
Up to now, 35 AA residues have been identified to affect thermostability of GUS (summarized in Supplementary Material, Table S2). Many residues have not been characterized in detail regarding their role in GUS protein's thermostability. We believe that some of these 35 AA residues may play an essential role while others may have minor roles in the GUS thermostability. As we have shown in this study, six non-essential AA residues have been identified using site-directed mutagenesis. This strategy can also determine whether mutations at these AAs are additive or interactive in altering thermostability facilitating further gene modification through rational evolutionary design (Xiong et al., 2006a).
Two new, previously not reported residues, Q493R and T509A, are also the key residues for the thermostability of GUS (Supplementary Material, Table S2). Based on the higher alanine residue content in thermophilic proteins, it was suggested that alanine residue was the best helix-forming residue (Argos et al., 1979). Facchiano et al. (1998) observed that helices of thermophilic proteins were generally more stable than those of mesophilic proteins. Some hyperthermophiles also show a significant decrease in glutamine residue in their proteins (Vieille and Zeikus, 2004), but the mechanism is currently unknown. The GUS-WT shares the same AA residues at 509(T), 550(N) and N566(N) with the human GUS. The GUS-TR3337 was mutated at these sites into 509(A), 550(S) and 566(S). At AA residue 559, GUS-WT has a G residue, while GUS-TR3337 and human GUS have S residues (Table 4). Overall, GUS-WT and GUS-TR3337 proteins show similar structural model as the human GUS protein does (Fig. 5A and B).
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Our results show that the six key AA residues of GUS-TR3337, M532T, G559S and N566S lie at tetramer interfaces, while the N550S is a buried residue. The residue of Q493R lies in the TIM-barrel domain near the catalytic residues, and at the tetramer interface. The residue of T509A was found near the active site in the crystal structure of the homologous human GUS. The residues Q493R, T509A, M532T, N550S, G559S and N566S were key points, together contributing to their high thermostability (Supplementary Material, Fig. S2). DNA shuffling experiments sometimes showed that AA changes distant from the active site can affect substrate specificity, improve kinetics or product specificities (Matsumura et al., 1999
; Flores and Ellington, 2002; Rowe et al., 2003). Whereas, the more conventional view is that AA changes near the active site are more likely to modify characteristics (Morley and Kazlauskas, 2005). Comparative analyses of the genomes of thermophilic and mesophilic organisms have previously revealed that thermophilic enzymes show marked preferences for some AA residues, especially those on the surface (Cambillau and Claverie, 2000; Fukuchi and Nishikawa, 2001). We found not only some surface AA residues but also some buried AA residues play key roles in maintaining the thermostability of GUS. In addition, all six key residues lie at the C-terminal and belong to the domain III, or the TIM-barrel domain, which contains the catalytic residues. These residues lie at the tetramer interface, and are located in a large cleft that occurs at the interface of two monomers. Using site-directed mutagenesis analysis, three AAs, D436E, V446A and A451V, were found not to play key roles in the thermostability GUS.
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Footnotes |
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Edited by Kam-Bo Wong
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Acknowledgments |
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This research was supported by Shanghai Rising-Star Program (05QMX1445); National and Shanghai Natural Science Foundation (30471258, 04ZR14116); 863 Program (2006AA10Z117, 06Z358); Shanghai Project for ISTC (055407068); NAFC Program (2006GB2C000086).
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Received January 14, 2007; revised April 29, 2007; accepted April 29, 2007.
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