Mandelamide Hydrolase: Structural Studies of a Novel Amidase
Master’s Thesis
Presented to Biochemistry Department Brandeis University
Dagmar Ringe, Advisor
In Partial Fulfillment of the Requirements for the Degree
Master of Science
By Matthew Hope
May 2009
Copyright by
Matthew Hope
2009
Acknowledgments:
The author would like to thank Dr. Jackie Naffin, Prof. Dagmar Ringe, Prof. Greg Petsko and all the members of the Petsko-Ringe Lab for their help, insight, and support.
iii ABSTRACT Mandelamide Hydrolase: Structural Studies of a Novel Amidase A thesis presented to the Biochemistry Department Graduate School of Arts and Sciences Brandeis University Waltham, Massachusetts By Matthew Hope
Mandelamide hydrolase(MAH), the first enzyme in the mandelamide pathway, catalyzes the
conversion of mandelamide in Psuedomonas putida which allows the species to grow without
gluclose. MAH has not been structurally characterized to atomic resolution yet, and doing so
would yield valuable information for studying the ways in which simple species evolve new
functions through mutagenesis. In this study, we explored three methods in stabilizing MAH to
in order to better facilitate crystallization and obtain a 3D-structure. Constructs of the enzyme
were rationally designed from a homology model to test the effects of foreshortening the amino
acid sequence. Also, dynamic light scattering (DLS) experiments were employed to find
conditions that will monodisperse laser light, indicating that the protein is more likely to
crystallize than aggregate. Finally, thermal denaturation curves were performed under various
conditions to find ways to thermally stabilize the enzyme in order to minimize unfolding and aggregation and promote crystallization. Although this inquiry did not yield crystals of MAH,
DLS conditions containing dithiothreitol (DTT) were found to be beneficial for minimizing
polydispersion and the thermal denaturation curves indicated that 6-dimethyl-4-heptyl-β-D-
maltoside improves the thermal stability. Qualitatively, foreshortening the protein also improves
the quality of crystallization drops. These results can be utilized in the future to stabilize MAH
and elucidate the proper crystallization conditions that could eventually lead to a structure at
atomic resolution.
iv Table of Contents: Page Number Title Page i Acknowledgments iii Abstract iv Table of Contents v List of Figures and Tables vi Introduction 1 Materials and Methods 7 Results and Discussion 11 Cloning, Purification, and Characterization of MAH 11 Dynamic Light Scattering Experiments 17 Melting Curves of MAH 22 Conclusion 32 References 33
v List of Figures and Tables:
Fig. 1: The Mandelamide Pathway in Psuedomonas putida, and the Lactamide Pathway Page 3 Table 1: Primers designs of MAHwt, MAHΔ7, and MAHΔ14 for PCR Page 7 Figure 2: SDS-PAGE Gels of MAH Purification Page 12 Figure 3: Activity of MAHwt and MAHΔ7 as Determined by Coupled Assay to L-Glutamate Dehydrogenase Page 13 Table 2: Kinetic Parameters of MAHwt, MAHΔ7, and MAHΔ14 Page 14 Figure 4: Circular Dichroism Standard Curves Page 15 Figure 5: Circular Dichroism Spectra of MAHwt, MAHΔ7, and MAHΔ14 Page 16 Table 3: DLS Results of Buffer-pH Screen for MAHwt Page 18 Table 4: Effect of Construct on MAH Polydispersion (MAHΔ7 vs. MAHwt) Page 19 Table 5: Effect of pH, Buffer and DTT Concentration on MAHwt Polydispersion Page 20 Figure 8: Standard Shapes for Protein Melting Curves with SYPRO-Orange Dye Page 23 Figure 9: Melting Curves of MAHwt with Increasing Concentrations of Protein and SYPRO Dye Page 24 Figure 10: Melting Curves of MAHwt with Increasing Concentrations of Protein and SYPRO Dye Page 25 Figure 11: Effects of F and Cl Inhibitor Concentration on MAHΔ7 Melting Curves Page 27 Table 6: Detergents from Hampton Screens 1-3 without SYPRO-Orange Dye Interaction Page 29 Figure 12: Effect of FOS-Choline 8 and 2,6-dimethyl-4-heptyl- β-D-maltoside on MAHwt and MAHΔ7 Fluorescence during Melting Curve Experiment Page 30
vi Introduction:
In order to live in harsh environments, organisms must adapt and alter their
biological functions to accommodate the evolutionary pressures of survival— this simple
idea was Darwin’s great insight into evolution. Biological science was irrevocably
changed after acquiring the theory of evolution through natural selection, but the
mechanisms of evolution on a cellular level are still a mystery. The ways in which simple
organisms, such as bacteria, acquire new chemical pathways or enzymes with new
functions is not well understood, and the ways that organisms alter their existing
machinery to regulate new functions is even more opaque. This project was undertaken as
part of a larger inquiry into the way that bacterial species incorporate foreign genetic
material into their own genome and acquire new abilities. The gain of new functions in
enzymes is of great interest to scientists, both for the knowledge of evolution on the sub-
cellular level as well as the potential applications. The ability to understand how
organisms adapt to new genetic material could lead to successful gene therapy, improved
drug resistance, genetically enhanced organisms, bioremediation, and a host of other
useful effects.
There are two main methods through which bacteria can acquire new functions:
mutations in existing enzymes and horizontal gene transfer of pathways into the organism
from outside. It has been shown that, on an evolutionary time scale, enzymes can relax
their substrate specificity and become mechanistically promiscuous through single amino
acid mutations. For instance, Schmidt et. al. showed that single mutations in the TIM barrel enzymes L-Ala-D/L-Glu epimerase (AEE) and muconate lactonizing enzyme II
(MLE) provided catalytic activity for the o-succinylbenzoate synthase reaction while
-1- maintaining wild-type function[1]. Therefore, duplication of enzymatic genes followed by random mutation seems to be a viable path to acquiring new functions in enzymes.
Horizontal gene transfer is another method of gaining functions (or even new pathways) that is routinely employed by biochemists, best exemplified by plasmid transformation.
[2,3,4]. A common example in nature is the transfer of antibiotic resistance among bacteria. The ways in which species transfer genetic material and then use the newly incorporated genes in conjunction with already existing pathways is not well understood.
The incorporation of any random pathway from another species could lead to up regulation or down regulation of other proteins, the creation of toxic byproducts, or changes in cellular regulation and transportation of proteins [5,6,7]. In order to study these effects in detail, the mandelamide pathway, a naturally occurring pathway found in
Psuedomones putida that allows for growth on mandelamide as the sole carbon source, was selected as a model system (see Fig. 1). To study gene transfer, E. coli will be used as a model system because the bacteria does not already have the mandelamide pathway, the metabolic pathways that E. coli does have are well characterized both enzymatically and transcriptionally [8], and it has been shown that products of the mandelamide pathway are non-toxic to E. coli.[9]
-2- Figure 1: The Mandelamide Pathway in Psuedomonas putida, and the Lactamide Pathway
The mandelamide pathway is noted in blue and converts R-mandelamide into benzoate, which then feeds into the β-ketoapidate pathway. Mandelamide hydrolase is the first enzyme in the pathway.Through directed mutagenesis, it is thought that the mandelamide pathway can be eventually converted into the lactamide pathway in E. coli, noted in red. By changing the specificity for substrate, this pathway would convert R-lactamide into acetate and then acetyl-CoA, which could be utilized by E. coli. [15 and Prof. Dagmar Ringe]
The mandelamide system was selected for study for several reasons. First, the
pathway is well characterized for all the enzymes listed in Fig. 1, although a few of the
enzymes do not have their structures solved to atomic resolution. Additionally, the
pathway is relatively diverse in the reactions it catalyzes in order to allow for a wide
range of processes to be studied. The mandelamide pathway also evolved recently which
suggests that specificity may not be completely hard-wired and that the pathway could be
used to study the effects of mutation [9]. The end product of the β-ketoadipate pathway
(which the mandelamide pathway feeds into) is catechol. Through the coupling of the
enzyme catechol 2,3-dioxygenase, catechol can be converted to 2-hydroxy muconate
-3- semialdehyde, whose yellow color can be used to easily monitor flux through the
pathway [10]. In addition, the enzymes of the mandelamide pathway could be mutated to
catalyze lactamide conversion into acetate, which would then feed into well- studied
metabolic pathways in E. coli, allowing for the examination of ways that new functions
that evolve through mutation are regulated in bacteria (see Fig. 1) [11,12]. If achieved, this would explore the second method of acquiring new activity in bacteria, enzyme mutation, and would provide the other half of the picture. The above factors make the mandelamide pathway a good model for studying the effects of incorporating genetic material from Pseudomonas putida into E. coli. Yet in order to completely elucidate the system, the structure of the first enzyme in the pathway, MAH, is required to atomic resolution. If the enzyme mutations are to be explored or designed in any rational way, a complete picture of the enzyme is required to assess the effects of site-directed mutation.
Therefore, obtaining a crystal structure of mandelamide hydrolase (MAH) was the main goal of this project.
The mandelate pathway consists of six enzymes to convert mandelamide into
catechol [13]. Some of these enzymes are well studied, such as mandelate racemase,
which was discovered to be evolutionarily similar to another TIM-barrel enzyme,
muconate lactonizing enzyme. This discovery prompted the idea of directed evolution
through site-directed mutagenesis. [14]. Currently, there are crystal structures of
mandelate racemase, S-mandelate dehydrogenase, and benzoylformate decarboxylase.
The mdlY gene product was proven to be mandelamide hydrolase(MAH) through
purification and activity assays [13]. MAH is a 54kDa monomeric enzyme that converts
mandelamide to mandelate with little enantiospecificity [15] hence it is the first protein in
-4- the proposed pathway. MAH also shows significant phenylacetamide activity, which
suggests the enzyme might be easily reengineered to accept other substrates besides mandelamide [15]. Through sequence alignment, it was discovered that MAH contains similarity to the amidase signature family, which has been shown to contain a novel Ser- cisSer- Lys catalytic triad [13]. The three members of the amidase signature family for which there are crystallographic data (malonamidase E2 (MAE2), fatty acid amide hydrolase (FAAH), and peptide amidase (PAM)) show near superimposable catalytic residues but very different contacts for specificity [16,17,18,19]. Verifying the presence of the novel triad as well as obtaining specificity information for mandelamide hydrolase through crystallographic determination was a major project goal.
Crystallization screening is the rate limiting step of many protein x-ray
crystallography inquiries. Varying temperature, pH, buffer, protein concentration, drop
size, and drop ratio alone allows for an overwhelming number of conditions to be tested,
and as such, it was decided to examine certain techniques in order to increase the
likelihood of discovering the right conditions. The three techniques employed to “screen
smarter” for crystallization conditions were rational design of constructs, dynamic light
scattering experiments, and high-throughput melting curves utilizing an RT-PCR
apparatus. We expect that these methods would provide clues as to which conditions to
explore that would stabilize the protein and facilitate crystallization. Upon acquiring a
sample of purified MAH with a His6-tag on the C-terminus from a previous study [15],
crystallization screening was attempted with little success. The mutant enzyme S204A
was also screened with the substrate bound, yielding fibers but not crystals of appropriate
size for diffraction [Unpublished]. A homology model constructed from other members
-5- of the amidase signature family showed that the C-terminal end of the enzyme appeared
to be unstructured [20]. It was decided to transfer the His-tag to the N-terminus of MAH
and produce two constructs of truncated length. It has been shown that changing
purification tags can aid in crystallization [21] as well as changing the terminus where the
tag is expressed [21]. Also, it could be that in solution, the unstructured C-terminus
prevents the enzyme from entering the packing structure correctly and that by shortening
the “tail”, the crystal contacts would be more favorable and larger crystals could form
[21]. Therefore, it was decided to clone two constructs truncated by seven amino acids and fourteen amino acids, MAHΔ7 and MAHΔ14, respectively. We expect that the reduction of the disordered C-terminus, along with DLS and screening with melting curves, will improve the likelihood of crystallization and lead to an atomic structure of
MAH.
In this study, the three constructs were cloned, expressed, and purified with high
yield. The DLS experiments yielded conditions where MAH is more monodispersed and less prone to aggregation. Also, screening with thermal denaturation curves revealed
conditions where MAH is stabilized by detergents. Crystal screening did not yield
crystals, but the frequency of disordered precipitate decreased significantly over the
course of the project due to utilizing the results of the experiments.
-6- Materials and Methods:
1. Materials The MAH sequence in a pet17 vector was obtained with permission from our collaborators, the McLeish group. Restriction enzymes, ligases, DNA polymerase, and other molecular biology reagents were obtained from New England Biolabs. PCR and sequencing primers were obtained from Operon. Unless otherwise stated, all other chemicals and reagents were purchased from either Fischer Scientific or Sigma Aldrich. 2. Preparation and Verification of the pet17mah vector The pet17mah vector was transformed into competent E. coli strain Dh5α cells and the cells were plated on LB medium with 0.1mg/mL ampicillin. Three single colonies were selected for growth in 5mL of LB and ampicillin. The plasmid DNA was extracted from the cultures using the QIAprep® Spin Miniprep Kit (QIAGEN). The vectors from the three strains were digested separately with the restriction enzymes HincII and SalI and run on an agarose gel. The expected basepair fragment size from HincII was 3261 and 1588bp, and the expected basepair fragment size for SalI was 4849bp. The gel showed that two of the strains had inserted the vector correctly, and one was selected for PCR. 3. Design of Primers and Polymerase Chain Reaction Three different constructs were designed: the full length, wild-type MAH (MAHwt), a construct that was truncated by seven amino acids at the C-terminus (MAHΔ7), and a construct truncated by fourteen amino acids at the C-terminus (MAHΔ14). On all three constructs, the His6 tag was expressed at the N-terminus of the MAH sequence. The pENTR Directional TOPO Cloning Kit (Invitrogen) and a Pfu Turbo DNA Polymerase were employed to insert the sequence into the gateway vector pENTR/D- TOPO. Primers were designed according to Table 1.
Table 1: Primers designs of MAHwt, MAHΔ7, and MAHΔ14 for PCR Construct Sequence Name Sequence MAH forward (wt, Δ7, Δ14) JN001MAH-GW-S1 5’- CACCATGCGCCACCC- AGTAGATATGCC-3’ MAH reverse wt JN002MAHfl-GW-AS1 5’- CTACATGGAAATTTT- GCTTTCAACCC-3’ MAH reverse Δ7 JN003MAHtr7¿GW-AS2 5’- CTACCCCGAACGCGT- CGGAGTCGGGCC-3’ MAH reverse Δ14 JN004MAHtr14¿GW-AS3 5’- CTAGCCACGCCCCAA- AATGCTCTCG-3’ Primers were designed for entry into the pENTR/D-TOPO vector (Invitrogen). The molecules were ordered from Operon.
The PCR gene products were run on an agarose gel and then gel purified. Purity was significantly increased.
-7- 4. Cloning using the pENTR Directional TOPO Cloning Kit The gene products were allowed to recombine with pENTR/D-TOPO vector. The entry vector was transformed into E. coli strain Dh5α cells and purified using the QIAprep® Spin Miniprep Kit (QIAGEN) in identical fashion to the above. The entry vector was allowed to recombine with destination vector pDEST17 using Clonase II Enzyme reaction. The destination vector was transformed into E. coli strain Dh5α and was extracted in an identical procedure as listed above. The fidelity of the MAHwt- His6, MAHΔ7-His6, and MAHΔ14-His6 plasmids was confirmed through sequencing (Genewiz). 5. Determining Conditions for Expression of MAHwt, MAHΔ7, and MAHΔ14 The expression of the three constructs proved problematic. Five strains of E. coli were used to find the best induction: BL21*, OrigamiII, MB1630, BL21pLysS, and MB1630pLysS, where pLysS is a plasmid that generates the enzyme lysozyme to degrade the cell wall of bacteria. The strains were transformed with the MAH constructs and a freshly transformed, single colony from each was used to inoculate 50mL of sterile LB broth with the appropriate antibiotics (BL21*: 5mg/mL ampicillin; OrigamiII: 5mg/mL ampicillin and 2.5mg/mL tetracycline; MB1630: 5mg/mL ampicillin and 5mg/mL spectinomycin; BL21pLysS: 5mg/mL ampicillin and 1.7mg/mL chloramphenicol; MB1630pLysS: 5mg/mL ampicillin, 5mg/mL spectinomycin, and 1.7mg/mL chloramphenicol) Cultures were grown at 37°C with vigorous shaking until the OD600 reached ~0.6-0.8 and then induced with 1M isopropyl β-D-thiogalactopyranoside (IPTG) at three different induction temperatures, 37°C, 25°C, and 18°C. The cells were harvested through centrifugation at 6,000rpm and 4°C and then lysed through sonication in 20mM potassium phosphate buffer pH7.0. The lysate was cleared though centrifugation at 10,000rpm and 4°C for 20min. Samples of lysate, supernatant, and pellet were run on an SDS-PAGE gel to compare levels of soluble and insoluble protein across cell lines and induction temperatures. Based on the results, the constructs were grown in batch with induction at 18°C in BL21pLysS for MAHwt and MAHΔ7, and MB1630pLysS for MAHΔ14. 6. Expression and Purification of MAHwt, MAHΔ7, and MAHΔ14 in Batch The constructs were purified in 6L batches. E. coli BL21pLys cells were transformed with the construct plasmid and plated on LB, ampicillin, and chloramphenicol plates (except for MAHΔ14, which was transformed into MB1630pLysS cells and plated on LB, ampicillin, spectinomycin, and chloramphenicol plates). A fresh, single colony was used to inoculate 50mL of sterile Luria broth with 5mg/mL ampicillin and 1.7mg/mL chloramphenicol which was grown overnight at 37°C with shaking. The MAH Δ14 culture also contained 5mg/mL spectinomycin. The cultures were used to inoculate 6L of Luria broth. The fresh cultures were grown at 37°C with vigorous shaking for several hours until the OD600 reached ~0.6-0.8, and were then induced with 1mM IPTG at 18°C. The cultures were allowed to incubate for approximately 2 days. Induction was checked using an SDS-PAGE gel. The cells were harvested through centrifugation at 6,000rpm and 4°C and were frozen. Upon thawing the pellets, lysis was achieved by sonication in 20mM potassium phosphate buffer pH7.0 along with one Complete EDTA-free Protease Inhibitor Cocktail Tablet (Roche). The lysate was cleared by centrifugation at 10,000rpm and 10°C for 20min and the supernatant was allowed to bind onto a Ni-NTA Agarose Column (QIAGEN)
-8- equilibrated with 20mM potassium phosphate buffer pH7.0 and 5mM imidazole (to prevent non-specific binding) for 2hr. 50mL fractions of 10, 20, 50, and 250mM imidazole were used to elute MAH off of the column and the fractions were pooled and collected. The fractions were dialyzed into 100mM HEPES, 1mM EDTA, and 5mM DTT buffer at pH7.0 overnight and then dialyzed again in the same buffer. The fractions were concentrated and stored at -80°C. 7. MAH Activity Assay MAH activity was determined at 25°C in 100mM potassium phosphate buffer with 1mM EDTA at pH7.8. The activity was measured through a coupled assay with L- glutamate dehydrogenase and α-ketoglutarate, measuring a decrease in absorbance of β-nicotinamide adenine dinucleotide phosphate (NADPH) at 340nm. Assays were performed in triplicate. A reaction mixture consisting of .8mM α-ketoglutarate, 15 units/mL L-glutamate dehydrogenase, .2mM NADPH, and 100mM potassium phosphate buffer was added to .02mg/mL MAH with varying concentrations (.1mM to 4mM) of mandelamide, the substrate. The total volume was 1mL for each reaction, and they were measured in UV-transparent cuvettes. Protein concentration was determined by a simple Bradford assay. Kinetic parameters were calculated by fitting the Michaelis-Menton equation using nonlinear regression analysis. 8. Circular Dichroism Spectropolarimetry Protein samples for far-UV circular dichroism (CD) were diluted into 20mM potassium phosphate buffer pH7.0 to 5μM for all three constructs. Far-UV CD spectra were recorded at 25°C for each construct, from 260nm to approximately 200nm at a pitch of .05nm and scanning speed of 1nm/min. The cuvette pathlength was 0.1cm and the instrument used was a Jasco J-810 Spectropolarimeter. Molar ellipticity was calculated according to the equation [θ] = θobs * 1 /(10lcn) where l = the pathlength in cm, c = the concentration of protein in M, and n = the number of amino acid residues (507). 9. Crystallization Screening with MAH Many different crystal conditions were set, although none yielded protein crystals. Crystal Screens 1-3 (Hampton), Wizard Screens 1-3 (Emerald Biosystems), Cryo Screen 1-2 (Emerald Biosystems), A/S Screen (Hampton), and the PEG Suite Screen (QIAGEN) were all tried. Approximately 2,300 conditions were sampled. The effects of changing construct, presence of inhibitors, protein concentration, drop size, and drop ratio were all explored, as well as numerous buffer compositions. Results from the DLS were also used to stabilize the protein buffer (100mM TRIS pH7.5 + 5mM DTT). The typical protein concentration was 10mg/mL with 3μL drops at a 1:1 ratio of protein and solution. All experiments were carried out at ambient temperature. 10. Dynamic Light Scattering Experiments DLS experiments to screen for conditions where the protein scatters laser light in a monodispersed fashion were carried out using a Protein Solutions DynaPro99. A simple 24 solutions screen was left over from a previous study. The screen varied buffer composition and pH; a crystal tray was set to test for precipitation, and all clear-drop solutions were tested with DLS. All DLS experiments were carried out at room temperature with 10mg/mL MAH in duplicate where possible. 15μL of buffer and 1μL of protein constituted one scan. The effects of buffer, pH, inhibitor,
-9- construct, and detergents were examined. If detergent was used, the cuvette was allowed to equilibrate for 5min before measurement. 11. Melting Curves using SYPRO-Orange Dye Thermal denaturation curves were determined for MAH using SYPRO-Orange Dye and a StepOnePlus Real Time PCR System by Applied Biosystems. Concentration of protein and dye, presence of inhibitors and detergents, and buffer conditions were all variables that were explored. Wells were loaded to a final volume of 25µL and then the plates were covered with optically transparent film, centrifuged at 2,000rpm for 30sec and then load into the machine. Temperature was increased from 25°C to 95°C in 0.3°C increments and data was processed and normalized using a Perl script written by Dr. Melissa Landon.
-10-
Results and Discussion:
The enzyme was cloned and purified in three forms: wt, Δ7, and Δ14. Expression and purification were followed by characterization in terms of activity and solution structure.
1. Cloning and Purification of MAHwt, MAHΔ7, MAHΔ14 All three constructs were successfully cloned from the pet17mah vector through the
use of the pENTR system. The expression and purification schemes yielded
crystallographic amounts of MAH in various levels of purity (see Fig. 2A,B,C).
Approximately 3mL of MAHwt at an average concentration of 10mg/mL were able to
be purified per 6L batch, and 1mL of MAHΔ7 and MAHΔ14 at an average
concentration of 16mg/mL was able to be purified per patch. The fractions were
pooled according to the concentration of imidazole, and then the imidazole was
dialyzed out. Wildtype MAH yielded the most protein, while the Δ7 and the Δ14
constructs were more difficult to purify in quantities as large as the wildtype.
-11- Figure 2: SDS-PAGE Gels of MAH Purification A. MAHwt B. MAHΔ7
C. MAHΔ14
Fig2A: Lane 1, molecular weight marker; Lane 2, cell lysate after sonication; Lane 3, supernatant after centrifugation; Lane 4, pellet after centrifugation; Lane 5, flowthrough on Ni column; Lane 6, 10mM imidazole fraction; Lane 7, 20mM imidazole fraction (first half); Lane 8, 20mM imidazole fraction (second half); Lane 9, 50mM imidazole fraction; Lane 10, 250mM imidazole fraction. Fig2B: Lane 1, molecular weight marker; Lane 2, cell lysate after sonication; Lane 3, supernatant after centrifugation; Lane 4, pellet after centrifugation; Lane 5, flowthrough on Ni column; Lane 6, 10mM imidazole fraction; Lane 7, 20mM imidazole fraction; Lane 8, 50mM imidazole fraction; Lane 9, 250mM imidazole fraction; Lane 10, empty. Fig2C: Lane 1, molecular weight marker; Lane 2, cell lysate after sonication; Lane 3, supernatant after centrifugation; Lane 4, pellet after centrifugation; Lane 5, flowthrough on Ni column; Lane 6 and 7, 10mM imidazole fraction; Lane 8, 20mM imidazole fraction; Lane 9, 50mM imidazole fraction; Lane 10, 250mM imidazole fraction.
-12- In addition, the activities of the constructs were assessed using a coupled assay to L- glutamate dehydrogenase, as described above (see Fig. 3A,B). Both the wildtype and the
Δ7 construct proved to be active, but the Δ14 did not show any activity, even at saturating
levels of substrate. Kinetic parameters were also determined (see Table 2).
Figure 3: Activity of MAHwt and MAHΔ7 as Determined by Coupled Assay to L- Glutamate Dehydrogenase A. MAHwt B. MAHΔ7
The activity assay for MAH wt and MAHΔ7 was carried out at 1mM protein in 100mM KPhosphate buffer pH7.8 with 1mM EDTA. The assay was performed at 25°C. Data was analyzed using the Kalidagraph program.
-13- Table 2: Kinetic Parameters of MAHwt, MAHΔ7, and MAHΔ14 -1 -1 -1 Kcat (s ) Km (μM) Kcat / Km (M s ) MAHwt 168 ± 9.5 130. ± 25 1.29 * 106 MAHΔ7 139 ± 10. 101 ± 28 1.38 * 106 MAHΔ14 n.d. n.d. n.d. MAHwt-His-tagged 26.4 ± 1.3 30.6 ± 3.4 8.62 * 105 The activity assays were carried out according to the method described above. “MAHwt-His-tagged” is the mandelamide hydrolase construct purified and characterized by McLeish et. al. [15] shown here for comparison. Note that the His6 tag is on the C-terminus for MAHwt-His-tagged and on the N-terminus for MAHwt. Finally, circular dichroism (CD) spectra were collected for the three constructs and compared to standard CD curves (see Figs. 4 and 5). The wild-type and Δ7 share similar spectra, while MAHΔ14 is slightly different. Both MAHwt and MAHΔ7 displayed two local minima: a narrow one at about 208nm and a wider one around 222nm. Both of these features are indicative of a typical spectrum of alpha helical character, but the widening of the 222nm minimum suggests there may also be some beta-sheet character, which would produce a minimum at 218nm. The MAHΔ14 spectrum is a wide shallow depression from about 225nm to 208nm which suggests that MAHΔ14 has a different fold from MAHwt and MAHΔ7, and some partial random coil character. The lack of observable activity and the altered CD spectrum is consistent with the hypothesis that truncating fourteen amino acids from the C-terminus prevents the construct from accessing the typical mandelamide hydrolase fold, or at the very least, greatly destabilizes the fold of MAHΔ14. MAHwt and MAHΔ7, however, appear suitable to use in crystallization procedures, which was the goal of their purification.
-14- Figure 4: Circular Dichroism Standard Curves
Standard circular dichroism spectra for α-helix β-sheet and random coil. Provided by www.proteinchemist.com.
-15- Figure 5: Circular Dichroism Spectra of MAHwt, MAHΔ7, and MAHΔ14 A. Non-Normalized Data
CD of MAH
20000
10000
0 185 190 195 200 205 210 215 220 225 230 235 240 245 250 255 260 265
-10000 Wildtype Delta7 Delta14 -20000 Molar Ellipticity
-30000
-40000
-50000 Wavelength (nm)
B. Normalized Data
Normalized CD Spectra of MAHContructs
1.2
1
0.8
0.6 Wildtype Delta7 Delta14 0.4
0.2 Normalized Fluoresence
0 185 195 205 215 225 235 245 255 265
-0.2 Wavelength (nm)
Spectra were determined at 5µM protein concentration as determined by absorbance spectrum using tryptophan residues. Data was normalized by setting maxima and minima to 1 and 0 respectively using the -1 equation: Normalized reading = 1 + [(v-vmax)/(vmin-v)] taken from Silva et. al. [23]
-16-
2. Dynamic Light Scattering Experiments The enzyme constructs were examined in terms of their aggregation potential in order to determine the best conditions for crystallographic trials. The way in which dilute protein solutions scatter laser light can give information about the relative stability of the protein in the solution in which it happens to be [22]. A protein that scatters light in a monomodal fashion with a low amount of polydispersion can be assumed to be freely rotating in solution and interacting elastically with other protein molecules [22]. If the scattering is elastic, then there is less of a chance of aggregation and it can be said that the interactions with the solvent are more favorable than the interactions with the other protein molecules. Low amounts of polydispersion correlates with the ability to crystallize proteins in sizes large enough for x-ray diffraction. Therefore, DLS techniques can be used to test different buffers for the relative stability they confer to proteins in order to select the buffer where the protein is most stable. The rationale behind this technique is to make the protein as stable as possible so that more control can be exerted when adding crystallization conditions, so that the protein is not too stable that it never crystallizes, but not overly unstable so that non-structured aggregates form.
Recommended polydispersion for good crystallization is in the low 20%s, and the expected hydrodynamic radius for MAH is about 7nm. High polydispersities and radii indicated significant aggregation. The first round of DLS experiments tested various buffers and pHs (see Table 3).
-17- Table 3: DLS Results of Buffer-pH Screen for MAHwt Buffer pH Peak Polydispersity (%) Est. Radius (nm) MOPS 7 52.3 104.8 MOPS 7 74.8 131.6 NH4OAc 7 44.7 111.2 NH4OAc 7 46.3 127.6 Phosphate 7 49.7 149.4 Phosphate 7 59.9 229 HEPES 7.5 53.1 102 HEPES 7.5 65.8 103.9 TRIS 7.5 63.6 104.8 TRIS 7.5 55.2 50.52 EPPS 8 52.1 78.41 EPPS 8 66.5 104 Imidazole 8 65.5 91.39 Imidazole 8 60.2 94.09 TRIS 8.5 43.9 70.7 TRIS 8.5 47.2 67.64 CHES 9 41.4 73.36 CHES 9 45.7 75.15 CHES 9.5 52.4 71.52 CAPS 10 45.3 75.93 DLS experiments were carried out at room temperature with 10mg/mL MAHwt, in duplicate. 15μL of buffer and 1μL of protein constituted one scan. Expected hydrodynamic radius of MAHwt is about 7nm. All buffers are 100mM.
From the results of this test it was decided to pursue NH4OAc, TRIS, HEPES, and CHES
at varying pH, because they had the lowest polydispersion. However, from the high
polydispersion and radius estimates, it is safe to conclude that MAHwt tends to aggregate in solution easily. It was decided to see if using a different construct would significantly
decrease polydispersion (see Table 4).
-18- Table 4: Effect of Construct on MAH Polydispersion (MAHΔ7 vs. MAHwt) Est. Difference in Average Radius Polydispersity from MAHwt Buffer pH Polydispersity (%) (nm) (%) MOPS 7 67 95.88 +3.4 NH4OAc 7 59.2 83.75 +13.7 HEPES 7.5 65.6 104.3 +6.2 TRIS 7.5 69.6 97.6 +10.2 EPPS 8 80.7 121.7 +21.4 Imidazole 8 68.4 95.13 +5.6 TRIS 8.5 58 72.58 +12.5 DLS experiments were carried out at room temperature with 10mg/mL MAHwt. 15μL of buffer and 1μL of protein constituted one scan. Expected hydrodynamic radius of MAHwt is about 7nm. All buffers are 100mM.
Switching to a shorter construct did not significantly decrease the polydispersion of the
solution, but in fact made it worse. Therefore it was decided to continue the experiments
exclusively with MAHwt. Taking the results from the first screen, the effects of altering
pH, buffer, and concentration of DTT were examined (see Table 5). The data from the
DLS experiments were highly variable, therefore it was decided to display the
polydispersity and radius readings unaveraged, so as to minimize the effect of outliers on
the numbers.
-19- Table 5: Effect of pH, Buffer and DTT Concentration on MAHwt Polydispersion Condition 0mM DTT 5mM DTT 10mM DTT Peak Peak Peak Peak Peak Peak 1 Peak 2 Peak 1 Peak 2 Peak 1 2 Poly- 1 Poly- 2 Poly- 1 Poly- 2 Poly- Poly- disper Radi dispe Radi dispe Radi dispe Radi dispe Peak1 dispe Peak2 sity us rsity us rsity us rsity us rsity Radius rsity Radius Buffer pH (%) (nm) (%) (nm) (%) (nm) (%) (nm) (%) (nm) (%) (nm) HEPES 7 63.2 106.1 72.0 132.5 73.0 43.68 HEPES 7 62.2 84.34 68.8 45.17 HEPES 7 75.6 139.2 50.4 32.48 25.9 297 HEPES 7.5 66.0 90.14 18.3 0.46 51.8 51.33 68.8 40.08 HEPES 7.5 74.0 107.5 43.8 28.47 HEPES 7.5 67.8 106.2 68.6 42.86 HEPES 7.8 71.5 101.7 56.0 91.84 58.8 37.84 HEPES 7.8 66.4 102.5 61.8 36.76 HEPES 7.8 68.9 97.85 55.0 32.15 15.5 8352 NH4OAc 7 49.6 100.9 68.4 118.9 57.8 35.84 NH4OAc 7 57.3 111.3 37.3 14.53 41.0 62.46 NH4OAc 7 66.2 118 59.1 32.19 NH4OAc 7.5 65.9 108.4 37.3 17.41 43.2 75.18 57.5 32.73 NH4OAc 7.5 60.3 99.52 46.5 26.39 17.9 309.1 NH4OAc 7.5 69.8 113.4 NH4OAc 7.8 46.8 74.52 94.1 67.18 53.4 30.65 NH4OAc 7.8 63.7 106.4 46.8 27.62 NH4OAc 7.8 65.6 109.6 NH4OAc 7.8 60.2 103 TRIS 7.5 59.8 54.57 13.9 3.7 51.3 43.25 11.6 3.56 51.9 43.65 17.3 3.12 TRIS 7.5 55.3 44.77 8.2 2644 59.9 50.17 52 47.54 11.5 3.61 TRIS 7.5 58.2 43.15 47.2 43.77 13.8 3.44 TRIS 7.5 62.7 52.41 TRIS 8 58.7 44.77 7.6 3.1 49.4 49.83 11.5 3.5 TRIS 8 61.6 52.26 3.2 2.95 49.1 44.16 9.2 3.0 TRIS 8 57.2 48.44 TRIS 8.5 63.1 50.15 5.4 2.8 78.9 78.52 54.6 48.81 11.0 2.81 TRIS 8.5 61.2 55.14 10.3 2600 44.8 42.44 52.1 52.82 TRIS 8.5 46.8 48.66 12.6 3.2 56.8 91.65 TRIS 8.5 42.2 39.32 5.5 2778 CHES 8.5 57.7 52.19 5.3 3.61 54.1 49.49 13.7 3.36 CHES 8.5 51.2 52.89 8.1 3.8 56.2 53.93 5.9 3.23 CHES 8.5 56.3 53.15 5.3 3.59 58.4 48.85 CHES 9 53 51.38 11 2.9 56.1 57.45 51.7 51.52 7.6 3.06 CHES 9 52.4 51.31 7.6 3.1 56.4 56.2 7.6 3.11 51.7 52.66 5.9 3.23 CHES 9 51.9 51.46 9.3 3 67.5 63.27 3.2 2.94 49.7 48.34 12.6 3.16 CHES 9 53.3 51.54 3.2 3 CHES 9.5 47.5 51.72 15.3 3.33 49.7 46.26
-20- CHES 9.5 54.3 55.33 5.3 3.61 46.8 44.35 4.3 4.37 CHES 9.5 54.7 50.54 51.8 45.46 DLS experiments were carried out at room temperature with 10mg/mL MAHwt, in triplicate where possible. 15μL of buffer and 1μL of protein constituted one scan. Expected hydrodynamic radius of MAHwt is about 7nm. All buffers are 100mM. Every pair of reading in bold is under 50% polydispersion.
Most of the hydrodynamic radii observed fell into three categories: between 3nm
and 7nm, 40nm and 50nm, or 100nm and above. These results suggest that the 3nm to
7nm measurements are of MAH in the non-aggregated form, because 3-7 is very close to
the expected hydrodynamic radius of a 54kDa protein (7nm). Additionally, the fact that
so many conditions gave peaks of about 50nm with large polydispersity suggests that
MAH also has a specific aggregate form that it greatly prefers to the non-aggregated form
under these conditions. Everything above 100nm seems to be non-specific aggregate that
can accumulate to almost any size, all the way up to 2600nm. The DLS experiments
proved that MAH has a strong tendency to aggregate, which explains why crystallization
is so difficult without stabilizing effects. Note also that most of the peaks with low
polydispersion are secondary peaks whose primary peaks have a polydispersion greater
than 50%, which means we are observing a system where there is a balance between non-
aggregated and aggregated forms of MAH. The area under the curves can also indicate
the amount of protein in each form, and simple observation indicates that there is always
more MAH in the aggregated form than the non-aggregated form under these conditions.
From the above results, it was decided to use 100mM TRIS pH7.5 with 5mM
DTT in screening for crystallization. We observed a trend that there are more conditions
with polydispersities under 50% in TRIS as opposed to any other buffer. Additionally, it
appears that there are more conditions with polydispersity under 50% for 5mM DTT as
opposed to the other concentrations. Most secondary peaks with low radii appear when
-21- DTT is added. MAH has an even number of cysteine residues, which does not
immediately suggest that reducing the thiol groups would prevent large-scale aggregation
since they should be in disulfides when folded properly, but nonetheless, DTT does have
a helpful effect on the polydispersity. TRIS pH 7.5 was selected because integration of
the peaks revealed that TRIS gave the largest area to the second peak, which suggests that
in TRIS, the largest amount of MAH will be non-aggregated in solution. MAH is
nowhere near the ideal polydispersity for crystallization under these conditions, which
suggests that crystallization is difficult because of a tendency to precipitate out of
solution. Qualitatively, using the buffer conditions obtained from the DLS experiments
increased the number of drops in crystallization that were clear or contained ordered
particulate, as opposed to drops obtained using HEPES buffer at pH 7.8. This suggests
that the buffer conditions have a stabilizing effect on the protein, which improves the
chances of being able to grow a suitable crystal.
3. Melting Curves of Mandelamide Hydrolase using SYPRO-Orange Dye Thermal denaturation curves were measured for MAH using a fluorescent dye in order to
screen for conditions that provide the greatest thermal stability to the enzyme. Stability in
solution is intrinsically related to thermal stability and can be measured using appropriate
spectroscopic markers. As temperature is increased, a more stable protein will unfold and denature at a slightly higher temperature than an unstable protein. This unfolding can be
measured by an increase in fluorescence of SYPRO dye, which interacts with
hydrophobic residues on the interior of the protein that only become exposed when the
protein is in the unfolded state. Through examining the relative temperatures of unfolding
-22- (Tm), it is possible to screen for stabilizing conditions. The higher the Tm, the more stable
the protein in solution and the better for crystallization.
For a protein melting curve, we expect to see an “S” shape (see Fig. 8). This is
expected when the protein starts off with a low level of fluorescence, increases the
fluorescent signal as it unfolds, and then decreases the signal as aggregation occurs and
the dye molecules are buried within protein aggregate. Alternatively, an aggregated or
unfolded protein will start off with a high signal and then decrease the signal as bleaching
or further aggregation occurs.
Figure 8: Standard Shapes for Protein Melting Curves with SYPRO-Orange Dye
The first set of experiments tested concentrations of protein and dye in order to maximize an orderly signal to continue screening (see Figs. 9 and 10).
-23-
-24-
-25- Both MAHwt and MAHΔ7 displayed more typical melting curve shapes as protein concentration increased (Figures 9 and 10, moving from A to D), with the most regular being found at 20µM, therefore 20µM was the protein concentration used for the rest of the screening process. Additionally, 20X dye seemed to give the least amount of decrease in signal for both constructs without sacrificing a low baseline of fluorescence, so it was also used for the rest of the screening process. Most importantly, both constructs are well behaved and give a good signal that can be studied using this method.
The effects of inhibitors for MAH obtained from the McLeish group were also examined using this method (see Fig. 11). The two inhibitors are 1-chloro-3-phenyl- propan-2one (Cl inhibitor) and 1,1,1-trifluoro-3-phenyl-propan-2-one (F inhibitor). The
Cl inhibitor has a Ki of 130µM and the F inhibitor has a Ki of 60µM.
-26- Figure 11: Effects of F and Cl Inhibitor Concentration on MAHΔ7 Melting Curves
20uM Protein, MAHDelta7
1.2
1
0.8 0mM F Inhibitor 05mM F Inhibitor. g 1mM F Inhibitor. 0.6 2mM F Inhibitor. 5mM F Inhibitor. Readin 1mM F Inhibitor 0.4 2mM F Inihbitor
0.2
0 0 1020304050607080 Temperature
20uM Protein, MAHDelta7
1.2
1
0.8 0mM Cl Inhibitor 05mM Cl Inhibitor. 10mM Cl Inhibitor. 0.6 2mM Cl Inhibitor. 5mM Cl Inhibitor. Reading 1mM Cl Inhibitor 0.4 2mM Cl Inhibitor
0.2
0 0 1020304050607080 Temperature
Denautration curves were performed in 100mM phosphate buffer pH 7.0. Fig.11A is the 1,1,1-trifluoro-3- phenyl-propan-2-one (F inhibitor) data, Fig.11B is the 1-chloro-3-phenyl-propan-2one (Cl inhibitor) data.
-27- The addition of inhibitors did little to alter the Tm of the protein. Additionally, the
F inhibitor appears to interact with the SYPRO dye at high concentrations yielding the
non-normal shapes pictured above. Control experiments with just the inhibitor and dye
confirm this (not pictured).
Finally, the effects of detergents on MAH were explored. Detergent screens 1 through 3 from Hampton with just potassium phosphate buffer and 20X dye were subjected to the melt curve procedure in order to determine which detergents interact significantly with the dye. Many detergents are at least partly hydrophobic, so significant interactions were expected, which would render the reading of a protein signal useless. 29 detergents did not interact with SYPRO dye (see Table 6), and those 29 detergents were used to screen MAHwt and MAHΔ7. Most curves displayed high fluorescence at low temperatures with decreasing fluorescence as the run continued, indicating aggregation at the start. However two detergents, FOS-Choline 8 and 2,6-dimethyl-4-heptyl-β-D- maltoside, appeared to stabilize the protein in the beginning stages and allow for an increase in signal, followed by a decrease in signal which is more reminiscent of a typical protein melting curve (see Fig. 12A and B).
-28- Table 6: Detergents from Hampton Screens 1-3 without SYPRO-Orange Dye Interaction Hampton Detergent Detergent Hampton Detergent Detergent Screen Number Screen Number Number Number 1 1 C12E9 3 3 n-Tetradecyl-Beta-D- maltoside 1 2 C12E8 3 4 n-Tridecyl-Beta-D- maltoside 1 3 n-Dodecyl-Beta-D- 3 6 ZWITTERGENT 3-14 maltoside 1 4 Sucrose monolaurate 3 7 n-Undecyl-Beta-D- maltoside 1 5 CYMAL-6 3 8 n-Decyl-Beta-D- thiomaltoside 1 7 CTAB 3 9 FOS-Choline 12 1 9 n-Decyl-Beta-D- 3 11 1-s-Nonyl-Beta-D- maltoside thioglucoside 1 15 DDAO 3 13 DDMAB 1 18 Heptyl-Beta-D- 3 14 n-Nonyl-Beta-D-maltoside thioglucoside 2 3 2,6-Dimethyl-4-heptyl- 3 17 FOS Choline 10 Beta-D-maltoside 2 14 IPTG 3 18 FOS Choline 9 2 15 n-Dodecyl-N,N- 3 21 FOS Choline 8 dimethylglycine 2 16 HEGA-10 3 23 ZWITTERGENT 3-08 2 24 C-HEGA-8 3 24 CYMAL-1 3 2 n-Hexadecyl-Beta-D- maltoside
-29- Figure 12: Effect of FOS-Choline 8 and 2,6-dimethyl-4-heptyl- β-D-maltoside on MAHwt and MAHΔ7 Fluorescence during Melting Curve Experiment A. MAHwt
B. MAH Δ7
Fig.16A:The green line represents the signal from protein with FOS-Choline 8 while the orange line represents the signal from protein with 2,6-dimethyl-4-heptyl-β-D-maltoside. The purple line is the control without any protein. Both detergents were selected because they show an increase followed by a decrease in fluorescence, indicating measurable unfolding. Fig. 16B: The green line represent the signal from protein with FOS-Choline 8 while the blue line represents the signal from protein with 2,6-dimethyl-4-heptyl-β-D-maltoside. The purple line is the control without any protein. Both detergents were selected because they display an increase followed by a decrease in fluorescence, indicating measurable unfolding.
-30-
It is possible that the two detergents help to stabilize MAH by interacting with exposed hydrophobic residues and making the protein more soluble in water. This would then allow the protein to unfold properly and give a more “S” shaped melting curve.
-31- Conclusions:
A crystal structure was not obtained during the course of this investigation, but the results of the inquiry can be utilized to make further crystallization trials more likely to be successful. Screening various conditions revealed ways that the protein can be made more monodispersed in solution and more thermally stable. Changing the construct did not significantly alter the results of the DLS experiments, but adding DTT and altering buffer and pH decreased polydispersity a moderate amount, suggesting that the protein is able to sample the non-aggregated state more under these conditions. Furthermore, detergent screening with thermal denaturation curves revealed two detergents that have significant stabilizing interactions with MAH which could potentially aid in crystallization. Further experiments would test the effect of these detergents on the success of crystallization screening. In addition, new constructs would be designed to test the point at which truncation causes inactivity as in the case of MAHΔ14, in order to create a less disordered construct that still maintains activity. Qualitative assessment shows that MAHΔ7 performs slightly better than MAHwt in crystallization trials, and an active, shorter construct might perform even better than MAHΔ7. With a little luck, a combination of the results from this study coupled with further crystallization trials could yield crystals that are suitable for diffraction and structural determination.
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-34-