CHAPTER 7 CRYSTALLIZATION AND CRYSTAL STRUCTURE STUDIES OF PGA

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1 CHAPTER 7 CRYSTALLIZATION AND CRYSTAL STRUCTURE STUDIES OF PGA 7.. Introduction This chapter describes the crystallization and structural studies attempted on two penicillin acylases KcPGA and AfPGA. Because of the limited yield of pure protein in the case of KcPGA only some preliminary crystallization experiments could be completed and we were not able to proceed with the structure analysis. However, the cloning and over production of this as an extracellular enzyme has been completed as described in chapter 6. Diffraction data at a resolution of 3.5 Å were collected from tetragonal crystals of AfPGA. The crystal structure was determined using molecular replacement method using coordinates of E. coli PGA (PDB:gk9). Although the resolution was modest we could identify features like the presence of a disulphide bridge in the structure that would be imparting comparatively more stability to AfPGA. The PGA from A. faecalis was cloned and characterized by Verhaert et al., 997. Despite its many similarities with other PGAs, the AfPGA enzyme has a clear industrial advantage over other well characterized penicillin acylases in βlactam conversions because of its higher thermostability and also for its high synthetic efficiency in enatioselective synthesis. This makes the AfPGA enzyme a more attractive biocatalyst both in hydrolysis and in synthetic conversions. The presence of two cysteine residues at positions B492 and B525 in AfPGA enzyme probably accounts for its higher stability on the basis of the following observations: (i) a comparison with the known 3D structure of the E. coli enzyme indicates that these two cysteine residues are in close proximity, (ii) the βsubunit of A. faecalis enzyme shows an electrophoretic mobility that depends on its oxidation state, (iii) the A. faecalis enzyme is significantly more thermostable in its oxidized state than reduced state, and (iv) AfPGA is more stable than the E. coli enzyme, which lack the disulfide bridge. These four arguments led us to conclude that a disulfide bond contributes for its higher stability compared to other PGAs. 223

2 7.2. Crystallization of KcPGA The attempts to obtain crystals using purified protein and commercial crystal screens were not successful. In many cases heavy precipitation occurred but failed to develop into crystals even after modifying the crystallization conditions. To try different strategy crystallization using different precipitants at ph 3 to 0 was explored. Small crystals appeared after 20 days in 40% ammonium sulphate in ph 8 and 8.5 of 50 mm MOPS buffer. The crystal size was improved by the addition of different concentrations of boctyl glucopyranoside and calcium chloride. The amount of protein used for the crystallization was 30 mg/ml. The size of the crystals were improved in 45% saturated AS, 60 µl 0.5% boctyl glucopyranoside and 50 mm CaCl2 in 50 mm MOPS buffer ph 8.2 grown at 303K (Figure 7.). These crystals were used for data collection both at room temperature and in liquid nitrogen temperature. The diffraction quality of the crystals was very poor at room temperature. A wide range of cryoconditions was screened. But, unfortunately the data collected at low temperature could not be indexed due to high mosaicity of the crystal. Attempts to collect good quality data using different cryoprotectants did not yield positive results. Subsequent attempts to improve the crystal quality by further altering crystallization conditions were also ineffective. Figure 7.: Crystals of KcPGA grown at 30 C. 224

3 7.3. Crystallization of AfPGA The purified protein sample was obtained from Dr. Zoya Ignatova (Institute of biotechnology, Technical University, Hamburg, Germany). This sample was checked in the SDSPAGE. The presence of β (64 kda) and α (20 kda) subunits were confirmed (Figure 7.2). A M β α Figure 7.2. SDSPAGE of purified AfPGA. The lanes contained A: AfPGA, M: markers Protein preparation The protein sample was dialyzed overnight with two changes against 00 times volume of 0 mm potassium phosphate buffer ph 7.5. Protein concentration was determined in accordance with the method of Lowry et al. (95) with BSA as standard. The protein was concentrated approximately 5 mg/ml in centricon concentrator (Millipore) at 5000xg rpm and was stored at 20 C. The protein sample used for crystallization was thawed and spun at 0000xg rpm for 5 min before keeping for crystallization Crystallization trials Crystallization conditions were initially screened using conventional sparsematrix screen with the commercial screening kits, Crystal Screens I and II (Hampton Research) and Clear Strategy screen I and II (Molecular Dimensions Ltd.). The screening 225

4 kits contained preparedready solutions and the crystallization trials were setup in hanging drop vapor diffusion technique. But unfortunately both the screens did not yield any crystals. The crystallization trials were setup using solutions of precipitant, salts and buffers. The screens were tested in a range of conditions where two parameters were varied, typically the precipitant concentration and the ph. In these trials the crystals were obtained primarily from two conditions (Figure 7.3). The fine screening for precipitation concentration and ph was carried out based on the initial observations. A number of additives were also tried to improve crystal quality. Finally, reproducible and diffraction quality crystals were obtained in 5% PEG 8000, boctylglucopyranoside (0.05 w/v) and 0. M TrisHCl ph 7.5. A number of crystal forms were obtained depending upon small variations in boctylglucopyranoside concentration. Unfortunately, none of the crystal forms diffracted under cryo conditions in the presence of different cryoprotectants or different strategies of soaking in cryosolution. The results are provided in Table 7. and Figure 7.4. Table 7.: Summary of cryoprotectant solution tried for AfPAG crystals S. NO Precipitant Cryoprotectant PEG 8K 25% PEG 400 No diffraction 2 PEG 0K 25 % PEG 400 No diffraction 3 PEG 0 K 30 % glycerol No diffraction 4 PEG 8K 30 % glycerol Around 8 5 PEG 8K 20%Ethylene glycol Poor diffraction 6 PEG 8K 20% hexane triol No diffraction 7 PEG 8K 20% isopropanol Around 5 8 PEG 0K 20%Ethylene glycol No diffraction 9 PEG 0K 20% hexane triol No diffraction 0 PEG 0K 20% isopropanol No diffraction 226 Result

5 a b c d Figure 7.3: Improvement in the quality and size of AfPGA crystals, a) Crystals grown in initial conditions with heavy amorphous precipitate, b) Monoclinic crystals grown at room temperature, c) Orthorhombic crystals obtained in the presence of boctyl glucopyranoside, d) tetragonal crystals 227

6 Data collection Considering the deterioration in quality of crystals irrespective of the cryosolution tried (Figure 7.4). The diffraction data were collected at room temperature by mounting the crystals in thin glass capillaries of 0.7,.0 or.5 mm diameter. Many crystal morphologies were observed and mostly turned out to be monoclinic forms (Table 7.2). Unfortunately in most crystal forms the completion of data collected was poor. Interestingly, some crystal transformation was taking place when the crystal was exposed to the Xrays. The orthorhombic P222 form has transformed into orthorhombic C222 form after certain period of exposure in Xrays (Figure 7.5). This type of cell transformation was already reported in lysozyme crystals (Harata & Akiba, 2006). The dehydration of lysozyme crystals induces molecular rearrangement, which transforms the crystals into a new crystals that is stable and has lower solvent content. The unit cell parameters of the both forms are in Table 7.2. Only in tetragonal form (P4) we were able to collect complete data. The details of data collection statistics were presented in Table.7.3. Because of one large unit cell dimension (298 Å) and not too excellent quality of the crystals we could collect data to an effective resolution of only 3.5 Å using the laboratory Xray source. a b Figure 7.4: The diffraction image of the AfPGA crystals collected in different cryoconditions a) 30% glycerol, b) 20% isopropanol 228

7 Table 7.2: Details of the different types of crystal forms of AfPGA characterized. S. no Crystal form Unit cell Mathew s Solvent No of molecules parameters number content in the (%) asymmetric unit ( and º) Monoclinic a=38.30 P2 b=7.65 c= β= Monoclinic a=75.25 P2 b=64.66 c=02.87 β = Monoclinic a=73.03 C2 b=86.80 c=262. β = Orthorhombic a=72.6 P222 b=86.7 c= Orthorhombic a=72.40 C222 b=86.02 c= Tetragonal P4 a=b= c=

8 a b c Figure 7.5: The indexed diffraction frames of C222 crystals to show the unit cell transformation during data collection a) spots are indexed in C222, b) many spots are not indexed after first 85 frames, c) from 95th frame the spots could be indexed in P

9 Table 7.3: Data collection statistics of the tetragonal crystal form of AfPGA Temperature Room temperature (RT) Wavelength (Å).548 Resolution (Å) ( ) Total reflections Unique reflections Completeness (%) 90.5 (95.4) R merge (%) 9.9 (23.9) Average I/s(I) 7.42 (3.08) Mosaicity 0.3 Space group P Unit cell parameters (Å) a=b=85.56 c= Solvent content (%) 6.3 Matthews Coefficient (Å3 Da ) 3.8 Molecules per Asymmetric unit 23

10 Structure solution Reflections were phased using molecular replacement method. The processed form of E. coli Penicillin G acylase monomer (PDB:gk9) was used as search model. The molecular replacement solution was obtained from PHASER. The Zscore and Rfactor indicated the correct solution. The P422 indexed data were used for the search. All possible alternative space groups (P 4 2 2, P 4 2 2, P 4 2 2, P 4 2 2, P , P , P , P ) were checked using the program in the calculation of translation function(table 7.4). The solution that corresponded to highest Zscore obtained (P4 2 2) was then subjected to packing function calculation with permissible number of clashes. In this case the permissible number was fixed as zero. The first cycle of refinement using Refmac was then carried out. Table 7.4: Fast Translation Function Table by Phaser. Space Group P422 SET TRIAL Top (Z) Second (Z) Third (Z) ( 7.0) ( 6.32) ( 6.06) P ( 5.94) ( 5.45) ( 5.34) P (3.60) (2.82) 4.28 (2.62) P (24.99) P ( 7.48) ( 6.53) 0.80 ( 6.08) P ( 6.06) 0.00 ( 6.02) ( 5.83) P (0.94) P (2.02) 45.0 (.34) 2.48 (0.49) Inspection of the electron density map after the first cycle of refinement has indicated that some disorder exists in the loop regions. For three loops electron density was completely absent. Ten more cycles of manual model fitting and refinement were carried out in conjunction with calculation of a full series of Quanta omit maps that were carefully examined for incorrect modeling. However, after a few cycles of refinement the 232

11 geometry of the model got totally distorted. The R factor reduced to not better than 25%, presumably due to poor quality of diffraction data Description of the preliminary structure of AfPGA The two chains of the molecule (α and β) are closely intertwined and form a pyramidal structure that contains active site in a deep coneshaped depression at the bottom. Among the 738 residues in the PGA, all but the eight Cterminal residues of the αsubunit could be modeled. The heterodimer has approximate dimension of 70 X 50 X 55 and has no obvious discrete domains (Figure 7.6). Figure 7.6: Preliminary structure of AfPGA showing heterodimeric association and αββα arrangement 233

12 Table 7.5: Comparison of sequence identity between two PGAs discussed in this thesis and EcPGA which is used as the model. EcPGA AfPGA KcPGA Total number of residues % homology with E. coli 4 86 No. of residues in αchain % homology with E. coli No. of residues in βchain % homology with E. coli No. of residues in spacer peptide % homology with E. coli The percentage of homology between the AfPGA and EcPGA is provided in table 7.5. From the sequence alignment using ClustalW, it is observed that the majority of residues in αchain are highly conserved in AfPGA(Figure 7.7), same as in the core of the protein, which includes the acyl cleft of the active site. The residues identified as directly involved in catalysis (Ser β, Ala β69, Asn β24, and Arg β263) are also strictly conserved. The peripheral region of the protein is the most variable one and the changes can be described as.four extra residues are present in AfPGA between amino acids α57 and α70 of EcPGA 2. The Cterminal portion of the αchain in AfPGA has two extra residues compared to EcPGA 3. Two residues in the AfPGA sequence are not present between residues numbers β332 and β337 of EcPGA. Together the α and βsubunits make up five distinct structural regions: an αβsandwich, two predominantly helical regions, a partial βbarrel, and a small βstrand region. Of the five structural regions, the largest is the βsandwich. This region contains 234

13 the catalytic residue Ser β and most residues of the hydrophobic specificity pocket. Surrounding and on either side of the core βsandwich region are the other four regions. 235

14 Figure 7.7: Alignment of PGA sequences discussed here to show the positions of conserved residues. A mostly helical region is situated on the sixstranded face of the bsandwich. It is composed of residues from the βsubunit and two α helices containing residues from the Cterminal end of the αsubunit. An entirely αhelical region, which contains majority of residues from the αsubunit, forms an interesting cluster of eight α helices. The disulphide bond was observed between the two regions β492 β525. (Figure 7.8). The electron density for this disulphide bond is clearly seen in the preliminary structure reported here AfPGA. 236

15 Figure 7.8: Disulphide bond in AfPGA shown along with (2FoFc) electron density map (σ) Figure 7.9: The superposition of Cα backbone of EcPGA (blue) and AfPGA (red) 237

16 The superposition of preliminary structure of AfPGA on EcPGA shows there is major deviation in the loop regions especially from residue number β360 to β370 and residue number β465 to β474. There are four key residues conserved in the active site of the AfPGA and EcPGA structures: the catalytic nucleophile residue Ser β, plus Gln β23, Ala β69, and Asn β24. The amide bond of the substrate is cleaved when the nucleophilic Oγ of Ser β attacks the carbonyl oxygen of the substrate forming an acylenzyme intermediate. Bond breakage is facilitated by positioning the substrate sidechain carbonyl group in oxyanion hole formed by the mainchain amide nitrogen of Ala β69 and Nδ of Asn β24 (Duggleby et al., 995). Most of the structural differences between the acylases EcPGA and AfPGA are located in the aminic subsite. This difference is mainly due to the mutation of Phe β7 in EcPGA to Pro in AfPGA. 238