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Bacillus licheniformis l-arabinose isomerase (l-AI) is distinguished from other l-AIs by its high degree of substrate specificity for l-arabinose and its high turnover rate. A systematic strategy that included a sequence alignment-based first screening of residues and a homology model-based second screening, followed by site-directed mutagenesis to alter individual screened residues, was used to study the molecular determinants for the catalytic efficiency of B. licheniformis l-AI. One conserved amino acid, Y333, in the substrate binding pocket of the wild-type B. licheniformis l-AI was identified as an important residue affecting the catalytic efficiency of B. licheniformis l-AI. Further insights into the function of residue Y333 were obtained by replacing it with other aromatic, nonpolar hydrophobic amino acids or polar amino acids. Replacing Y333 with the aromatic amino acid Phe did not alter catalytic efficiency toward l-arabinose. In contrast, the activities of mutants containing a hydrophobic amino acid (Ala, Val, or Leu) at position 333 decreased as the size of the hydrophobic side chain of the amino acid decreased. However, mutants containing hydrophilic and charged amino acids, such as Asp, Glu, and Lys, showed almost no activity with l-arabinose. These data and a molecular dynamics simulation suggest that Y333 is involved in the catalytic efficiency of B. licheniformis l-AI.
l-Arabinose isomerase (l-AI) is an enzyme that mediates in vivo isomerization between l-arabinose and l-ribulose as well as in vitro isomerization of d-galactose and d-tagatose (20). l-Ribulose (l-erythro-pentulose) is a rare and expensive ketopentose sugar (1) that can be used as a precursor for the production of other rare sugars of high market value, such as l-ribose. Despite being a common metabolic intermediate in different organisms, l-ribulose is scarce in nature. The market for rare and unnatural sugars has been growing, especially in the sweetener and pharmaceutical industries. For example, several modified nucleosides derived from l-sugars have been shown to act as potent antiviral agents and are also useful in antigen therapy. Derivatives of rare sugars have also been used as agents against hepatitis B virus and human immunodeficiency virus (2, 22).
For these reasons, interest in the enzymology of rare sugars has also been increasing. Various forms of l-AI from a variety of organisms have been characterized, and some have shown potential for industrial use. Several highly thermotolerant enzyme forms from Thermotoga maritima (12), Thermotoga neapolitana (10), Bacillus stearothermophilus (18), Thermoanaerobacter mathranii (9), and Lactobacillus plantarum (5) have been characterized previously. All of these reported l-AIs tend to have broad specificity, although a few l-AIs with high degrees of substrate specificity for l-arabinose have also been documented.
The enzyme properties of l-AIs have been examined by engineering several forms by error-prone PCR and site-directed mutagenesis. Galactose conversion was reportedly enhanced 20% following site-directed introduction of a double mutation (C450S-N475K) into l-AI (16). Error-prone PCR manipulation of l-AI from Geobacillus stearothermophilus resulted in a shift in temperature specificity from 60 to 65°C and increased isomerization activity (11). All of these previously reported mutational studies have been aimed at improving enzymatic properties for industrial application. However, even though the three-dimensional (3D) structure of Escherichia coli l-AI has been determined previously (15), few new structural studies have been performed to decipher the reaction mechanism of this enzyme. Rhimi et al. (19) have reported an important role for D308, F329, E351, and H446 in catalysis, as indicated by findings from site-directed mutagenesis. Nonetheless, detailed analysis of the important molecular determinants controlling the catalytic activities of the l-AIs is still lacking.
Previously, we have reported the cloning and characterization of a novel l-AI from Bacillus licheniformis (17). This enzyme can be distinguished from other l-AIs by its wide pH range, high degree of substrate specificity for l-arabinose, and extremely high turnover rate. In the present paper, we report the identification of an important amino acid residue responsible for the catalytic efficiency of l-AIs, as determined by a systematic screening process composed of sequence alignment and molecular dynamics (MD) simulation, followed by site-directed mutagenesis. Using the crystal structure of E. coli l-AI as a template, we have built a 3D model of B. licheniformis l-AI. Analysis of the 3D model of B. licheniformis l-AI docked with l-arabinose, followed by a systematic screening process, showed that Y333 interacted with the substrate, suggesting that this residue in B. licheniformis l-AI may be essential for catalysis. We further characterized the role of Y333 in B. licheniformis l-AI binding of and catalytic efficiency for l-arabinose.
Reagents for PCR and Ex-Taq DNA polymerase were purchased from Promega (Madison, WI). Restriction enzymes were obtained from New England Biolabs (MA). The pQE-80L expression vector, a plasmid isolation kit, and an Ni-nitrilotriacetic acid Superflow column for purification were from Qiagen (Hilden, Germany). Oligonucleotide primers were obtained from Bioneer (Daejeon, South Korea). Electrophoresis reagents were from Bio-Rad, and all chemicals for assays were from Sigma-Aldrich (St. Louis, MO). A plasmid containing the wild-type B. licheniformis l-AI gene (17) was used for the production of wild-type B. licheniformis l-AI protein. The araA gene from E. coli W3110 was amplified by PCR using two oligonucleotide primers, 5′-CCGGAATTCATGACGATTTTTGATAATTATG-3′ (an EcoRI restriction site is underlined) and 5′-ATTACTCGAGGCGACGAAACCCGTAATAC-3′ (an XhoI restriction site is underlined). The araA gene released from the pGEM-T Easy vector (Promega) was ligated with the pET-28a vector to give pET-araA, in which araA is under the control of the T7 promoter. The cloned gene was confirmed to be free of point mutations by DNA sequencing. E. coli BL21(DE3) was transformed with the recombinant plasmid for protein expression. E. coli strains harboring wild-type and mutated B. licheniformis l-AI genes for protein expression were grown in Luria-Bertani (LB) medium supplemented with ampicillin (100 μg/ml) at 37°C. E. coli strains harboring wild-type and mutated genes for E. coli l-AI were grown in LB medium supplemented with kanamycin (50 μg/ml). Isopropyl-β-d-thiogalactopyranoside (IPTG) was then added to the culture medium at a final concentration of 0.5 mM, and incubation continued with shaking at 37°C.
Site-directed mutagenesis was carried out by using the QuikChange site-directed mutagenesis kit from Stratagene (La Jolla, CA). The recombinant plasmid pQE-araA (17) containing the wild-type l-AI gene was used as the DNA template. The plasmids containing the correct mutant genes were then used to transform E. coli BL21(DE3), and colonies selected by ampicillin resistance were used for protein expression.
Wild-type and mutant enzymes were purified by the same procedure. Cell pellets were suspended in 20 mM sodium phosphate buffer (pH 7.5). The cell suspension was incubated on ice for 30 min in the presence of 1 mg/ml lysozyme. Cell disruption was carried out by sonication at 4°C for 5 min, and the lysate was centrifuged at 14,000 × g for 20 min at 4°C to remove the cell debris. The resulting crude extract was retained for purification. The cell extract was applied to an Ni-nitrilotriacetic acid Superflow column (3.4 by 13.5 cm; Qiagen) previously equilibrated with a binding buffer (50 mM NaH2PO4, 300 mM NaCl, pH 8.0). Unbound proteins were washed out from the column with a washing buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 8.0). Then the protein was eluted from the column with an elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0). Enzyme fractions were analyzed by sodium dodecyl sulfate-12% polyacrylamide gel electrophoresis (SDS-12% PAGE) and visualized by staining with Coomassie blue R250. Protein concentrations were determined by the Bradford method using bovine serum albumin as a standard protein (3).
l-AI activity was measured by determination of the amount of l-ribulose formed. Under standard conditions, the reaction mixture contained 1 mM MnCl2, ~15 μg of enzyme, 250 mM l-arabinose (the substrate), and 20 mM phosphate buffer (pH 7.5) to bring the final volume to 100 μl. The reaction mixture was incubated at 50°C for 5 min and then cooled on ice to stop the reaction. The l-ribulose generated was evaluated by the cysteine-carbazole-sulfuric acid method, and the absorbance at 560 nm was measured (6). Kinetic parameters for B. licheniformis l-AI were determined by using a mixture of 20 mM phosphate buffer (pH 7.5), 1 mM Mn2+, and 1 to 1,200 mM substrate (l-arabinose). One unit of l-AI activity was defined as the amount of enzyme catalyzing the formation of 1 μmol ketosugar per min under the above-specified conditions.
The 3D homology models of the wild-type and all mutant proteins were generated using the Build Homology Models module in the MODELER application of Discovery Studio 2.1 (DS 2.1; Accelrys Software Inc., San Diego, CA). The crystal structure of E. coli l-AI (Protein Data Bank accession code 2ajtA) was used as a template. Comparative modeling was performed to generate the most probable structure of the query protein by alignment with template sequences, simultaneously satisfying spatial restraints and local molecular geometry. The fitness of the model sequences in the present 3D environment was evaluated by the Profile-3D Score/Verify Protein tool in MODELER as implemented in DS 2.1. A discrete optimized protein energy (DOPE) score in MODELER was also calculated to determine the quality of protein structures. The root mean square deviation (RMSD) between the models and the template was calculated by superimposing the models onto the template crystal structure. The evaluated 3D model was used for docking and postdocking analyses. Hydrogen atoms were first added to the 3D models, and then the added hydrogen atoms were minimized for stable energy conformation and relaxation of the conformation from close contacts. Different substrate molecules were docked into the substrate binding pockets (SBP) of B. licheniformis l-AI and a mutant model by using C-DOCKER, an MD simulation-annealing-based algorithm module from DS 2.1 (23). Different poses were then created using random rigid-body rotation followed by simulated annealing. Before docking, the structures of the protein, the substrate, and the corresponding complexes were subjected to energy minimization using a CHARMm (4) force field as implemented in DS 2.1. A full potential final minimization was then used to refine the substrate (ligand) poses. The substrate orientation which gave the lowest interaction energy was chosen for another round of docking. Based on the C-DOCKER energy, the docked conformation of the substrate was retrieved for postdocking analysis.
Optical spectra were recorded with a Cary 100 Bio UV-Vis spectrophotometer (Varian, Palo Alto, CA). Circular dichroism (CD) experiments were performed with a Jasco J-815 spectrophotometer at 20°C. Each spectrum was recorded in the 190- to 300-nm region at the rate of 100 nm/min. CD spectra were corrected with respect to the baseline, and the measured ellipticity for each sample was expressed in millidegrees. The amino acid sequences deduced from the araA gene sequences of B. licheniformis were compared with those of related enzymes from other sources by using the BLAST network at the National Center for Biotechnology Information. The multiple-sequence alignment was performed with the ClustalW program.
To locate the conserved residues in B. licheniformis l-AI, the amino acid sequence from B. licheniformis was aligned with other l-AI sequences from Bacillus halodurans, Lactobacillus lactis, Lactobacillus plantarum, Thermotoga maritima, E. coli, Klebsiella pneumoniae, Thermoanaerobacter mathranii, Thermotoga neapolitana, Bacillus stearothermophilus, Geobacillus stearothermophilus, Thermus sp., Geobacillus thermodenitrificans, and Bacillus subtilis. Multiple-sequence alignment of l-AIs from these organisms revealed 60 different amino acids, including the 4 active site residues (E306, E331, H348, and H447 in B. licheniformis l-AI), that were totally conserved (100% identical) throughout the sequences (see Fig. S1 in the supplemental material).
B. licheniformis l-AI had a level of sequence identity to E. coli l-AI of 50%. By taking advantage of the X-ray crystal structure of E. coli l-AI (Protein Data Bank entry 2ajtA) and molecular modeling, a homology model of B. licheniformis l-AI was constructed (Fig. (Fig.1A).1A). The generated model was then validated by Ramachandran plots (14). In the B. licheniformis l-AI model, 97.8% of residues were located within the allowed regions, with 91.8% of the residues in the favorable region and 6% of the residues in the remaining allowed region. Only 2.2% of the residues were located in the outlier regions of the Ramachandran plot. The Profile-3D score for the model was 175, versus the maximum expected score of 212. The constructed model was also evaluated by superimposing it onto the template crystal structure, and the RMSD between the model and the template was 0.51 Å based on C-α atoms.
The substrate l-arabinose was docked into the homology model using DS 2.1 software. A total of 34 amino acid residues, including the 4 active site residues, were found within 5 Å of the SBP (see Fig. S2 in the supplemental material). Putative active site residues of E. coli l-AI had previously been proposed to be E306, E333, H350, and H450 based on the crystal structure of E. coli l-fucose isomerase (21). Upon superimposition, residues E306, E331, H348, and H447 of B. licheniformis l-AI corresponded to the proposed putative catalytic residues (E306, E333, H350, and H450) of E. coli l-AI (Fig. (Fig.1B1B).
In order to confirm these findings, site-directed mutagenesis to change each of these four residues to Ala was performed. The E306A, E331A, H348A, and H447A mutant proteins had no measurable isomerase activity, supporting a role for the corresponding mutated residues as active site residues required for isomerization (15). Upon confirming the role of the 4 putative catalytic residues, we turned our focus to 12 other conserved residues within 5 Å of the SBP of B. licheniformis l-AI. The roles of 5 of these 12 conserved residues, F279, D308, F329, E351, and H446, have been described previously (19). The roles of the remaining seven residues (M185, T276, Y333, L345, M349, I370, and W439) were therefore investigated by further site-directed mutagenesis (Fig. (Fig.22).
To probe the functional roles of the selected conserved residues, all selected residues were individually mutated to Ala. The recombinant enzymes carrying an M349A, W439A, T276A, L345A, I370A, M185A, or Y333A mutation were expressed and purified (Fig. (Fig.3).3). When the activities of the mutants with l-arabinose were analyzed and compared with that of wild-type B. licheniformis l-AI, only the substitution at Y333 was found to cause any significant change in l-AI activity (data not shown). The specific activity of the Y333A mutant was determined to be 3 μmol/min/mg of protein for l-arabinose, which corresponded to 2.8% of that of the wild-type enzyme (105 μmol/min/mg of protein). This pronounced loss of activity indicated that Y333 in the SBP near the substrate significantly modulated the catalytic efficiency for the substrate. Thus, this residue was considered to be a crucial determinant of the catalytic efficiency of B. licheniformis l-AI. The role of position 333 in catalytic efficiency was therefore further investigated by thorough site-directed mutagenesis.
Tyr in position 333 was replaced with nonpolar aromatic, nonpolar hydrophobic, and polar/charged residues by site-directed mutagenesis. All mutants were expressed at a level similar to the wild type (17). All purified mutant proteins exhibited similar CD spectra, with ellipticity minima of comparable amplitudes in the 220- to 230-nm range (Fig. (Fig.4).4). This observation is a good indication that all enzymes were properly folded. When Y333 was replaced with polar, charged amino acids (yielding mutations Y333K, Y333E, and Y333D), the mutant enzymes showed no activity toward l-arabinose. When Y333 was replaced with nonpolar, aliphatic, and hydrophobic amino acids (generating Y333I, Y333V, and Y333A mutants), the activity with l-arabinose was significantly decreased. The specific activities of Y333I, Y333V, and Y333A mutants were 28, 18, and 3 μmol/min/mg of protein, respectively, which were 27, 17, and 2.8% of wild-type B. licheniformis l-AI activity, respectively.
To further investigate the role of the aromatic ring of Y333 in the active site, the residue was replaced with Phe or Trp. Neither of these replacements altered the activity significantly, as the activities of Y333F and Y333W mutants were 91 and 76 μmol/min/mg of protein, respectively, compared to that of the wild type of 105 μmol/min/mg of protein. This finding suggests that the aromatic ring of Tyr, Phe, or Trp is likely to be involved in binding the pyranosyl ring of l-arabinose. Site-directed mutagenesis in E. coli l-AI was performed to mutate Y335, which is the residue corresponding to B. licheniformis l-AI Y333, to Ala. The Y335A mutant exhibited significantly decreased l-AI activity toward l-arabinose (less than 2% of wild-type E. coli l-AI activity), indicating that the residue at position 335 is critical for the activity of E. coli l-AI.
The kinetic parameters determined for purified wild-type and mutant B. licheniformis l-AI enzymes acting on l-arabinose are shown in Table Table1.1. Among the mutants containing a nonpolar aliphatic residue, the decrease in catalytic efficiency (the kcat/Km ratio) was correlated with the decrease in the size of the amino acid side chain. Comparisons of the kinetic parameters for Y333 mutants to those for the wild type suggested that the maximum turnover rate and catalytic efficiency were maintained by aromatic amino acids at position 333 (Table (Table11).
Changes in Δ(ΔG) were determined based on kinetic parameters for the six generated mutant enzymes (Table (Table1).1). We then investigated the relationship between Δ(ΔG) and the SBP of B. licheniformis l-AI by analyzing the active site structures of the mutant B. licheniformis l-AI models. In order to investigate the interaction between the substrate and each amino acid residue that had been mutated in the B. licheniformis l-AI models, the distance between each residue and l-arabinose was calculated from the predicted model using MD simulation (Fig. (Fig.5).5). When compared to the wild-type B. licheniformis l-AI, the Y333I and Y333V mutants showed significant decreases in catalytic efficiency (to 4.0 and 2.8 min−1 mM−1, respectively) and increased Δ(ΔG) values (5.76 and 6.71 kJ mol−1, respectively), which were probably the result of the increased distance between the residue and the substrate. However, the Y333F (Fig. (Fig.5B)5B) and Y333W (Fig. (Fig.5C)5C) mutant enzymes showed no significant changes in catalytic efficiency.
B. licheniformis l-AI has been reported to be the l-AI with the highest turnover rate for l-arabinose (17). In this study, we used a systematic strategy to identify the molecular determinants of this catalytic efficiency: we screened for conserved residues by multiple-sequence alignment and then used MD simulation to identify conserved residues in contact with the substrate, followed by individual site-directed mutagenesis to change those residues. The molecular docking study and mutational analyses of residues in contact with the substrate in the SBP of the wild-type B. licheniformis l-AI showed significant interaction between the substrate and Y333. The Y333A mutant lost about 97% of the activity of the wild-type enzyme. Further mutations at position 333 indicated that an aromatic amino acid at position 333 is essential for the catalytic efficiency of B. licheniformis l-AI. A similar result was obtained with E. coli l-AI, chosen as another model l-AI enzyme. These results suggest that this position can be considered a crucial determinant for the catalytic efficiency of all l-AIs.
In the crystal structures of E. coli l-fucose isomerase and E. coli l-AI, C-1 and C-2 of the substrate have been shown to transfer protons via an enediol intermediate (15, 21). Isomerization between l-arabinose and l-ribulose is depicted in Fig. Fig.6.6. During the aldose-ketose interconversion, two hydrogen atoms are transferred via an enediol intermediate. The proton transfer is facilitated by two residues, Glu306 and Glu331, corresponding to the mechanism suggested for E. coli l-AI and E. coli l-fucose isomerase. Thus, the binding of C-1 and C-2 of the substrate to the catalytic residues gains importance for completion of the isomerization reaction. When l-arabinose was docked into the active site pocket of wild-type B. licheniformis l-AI, hydrogen bonding between C-1, C-2, and C-4 of the substrate and the E331, E306, and E331 residues, respectively, occurred, with bond lengths of 2.28, 2.07, and 2.3 Å, respectively (Fig. (Fig.5A).5A). In the Y333A mutant, there can be no H bonding because of the increased distance between C-1 and C-2 of the substrate and the active site residues E331 and E306 (Fig. (Fig.5D).5D). This increased distance in the Y333A mutant adequately explained the reduced specific activity of the Y333A mutant for the substrate. When Tyr was replaced with Phe, the kcat was retained at its maximum, as the H bonding between C-1, C-2, and C-4 of the substrate and the active site E331, E306, and E331 residues could be retained with the lengths of 2.1, 3.07, and 2.06 Å, respectively (Fig. (Fig.5B5B).
The turnover rates for the mutants containing nonpolar aliphatic residues were as follows: rate for the Y333I mutant > rate for the Y333V mutant > rate for the Y333A mutant. This pattern correlated with the decreasing size of the side chain and its decreasing hydrophobicity (Table (Table1).1). When Y333 was replaced with a charged polar amino acid such as Arg, Asp, or Glu, the mutants exhibited no l-AI activity because the charged amino acid disrupted the essential hydrophobic interaction with the substrate. These results demonstrate that position 333 requires an aromatic amino acid, which probably functions to position the arabinopyranosyl ring so that the important H bond interaction with the active site residues is possible. Indeed, analysis of the 3D models of the various mutants showed that a slight modification of the position of l-arabinose in the active site occurred, which induced a change in the distance between E306/E331 and the substrate. This resulted in less efficient isomerization of l-arabinose by the mutant B. licheniformis l-AIs than by wild-type B. licheniformis l-AI.
Previously reported X-ray crystal structures of rabbit phosphoglucose isomerase (13) and mouse phosphoglucose isomerase (7) with cyclic forms of the substrate that mimic the enediol intermediate led to proposed mechanisms for the ring-opening (or ring-closing) and isomerization steps in the multistep catalytic mechanism. Similar aldose-ketose isomerization mechanisms for yihS-encoded proteins from E. coli and Salmonella enterica (8) have been described previously. Strongly conserved aromatic amino acids (Trp51, Tyr111, Trp316, Phe329, and Trp375) in YihS protein were found to interact with the substrate (8). In the present study, our postdocking analysis suggests that the aromatic side chain of Y333 is involved in a similar stabilizing interaction of the protein-ligand complex (Fig. (Fig.5).5). Indeed, the Δ(ΔG) value for the wild-type B. licheniformis l-AI was much lower than the values determined for the mutant B. licheniformis l-AIs (Table (Table1).1). This was due probably to the stabilization of the protein-ligand complex by the interaction between the residue Y333 and l-arabinose. The function of the aromatic group of Y333 (or Trp or Phe) would be the constraint of reaction intermediates (chain forms) after ring opening to facilitate the following ring closure.
This work was supported by the 21C Frontier Microbial Genomics and Applications Center Program, Ministry of Education, Science and Technology, Republic of Korea. It was also supported by a grant (code 2008A0080126) from the Agricultural Research and Development Promotion Center.
Published ahead of print on 4 January 2010.
†Supplemental material for this article may be found at http://aem.asm.org/.