Bitirme projesi - ENZYME DESIGN-son

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1. INTRODUCTION

Enzymes are highly developed catalysts that make metabolism and life

possible.They are proteins , often functionalized with cofactors,that catalyze a wide variety

of reactions. [1]

Enzymes evolved over three-billion years can catalyze chemical transformations that

are too slow to be measured under normal conditions. The most proficient enzymes can

accelerate reactions with turnover rates that occurs rapidly as the diffusion of reactants to

the catalyst[1].

Humans have long taken advantage of enzymatic processes for many processes

involving natural fermentation , for examples to preserve foods, to make bread and cheese,

or to produce alcoholic beverages.Even though the identity of enzymes was not known

suggested at the time, Emil Fisher suggested in 1884 a lock an key model to explain the

substrate specifity of enzymes. In 1946, Linus Pauling suggested that enzymes are closely

complementary in structure to the activated complex fort he reaction catalyzed, a very

impressive speculation considering that at the time ‘no one succeeded in determining the

structure of any enzyme nor in finding out how the enzyme does its job’[2].

A surge of active research related to enzyme catalysis has continued to expand our

understanding ofthe natures extraordinary catalysts. A variety of factors have been

proposed to explain the observed rate enhancements, these range from noncovalent

transition state stabilization to covalent bonding [3]. Enzymes work with remarkable

catalytic proficiencies, which is formally the binding constant of the complex formed

between the enzyme and the transition state, as was defined by Wolfenden [3].

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Chemists imagine the possibility of designing and synthesizing molecules with the

attributes of enzymes. Furthermore, combining catalytic turnover with substrate-stereo-

regio-or chemoselectivity as well as a tolerance towards organic solvents, elevated

temperatures, and chemical degradation is highly desirable for industrial applications.

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Figure 1.1. Enzyme Design 4

The availability of powerful computers, and high performance computer austers

have led to the first succesful examples of designed enzymes that can accelerate the rates

of reactions that are not catalyzed by nature [5]. However,the designed enzymes show only

modest rate enhancements, and perform particularly poorly at binding the transition states

of reactions.That is,there are many more challenges to overcome in the area of

computational enzyme design. In this project,an alternative strategy to ‘inside-out’

approach was explored [5].

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1. THEORETICAL BACKGROUND

2.1.THE INSIDE-OUT APPROACH TO COMPUTATIONAL ENZYME DESIGN

Efficient computational algorithms have been developed for identifying amino acid

sequences compatible with a target tertiary structure. Much effort has been devoted to

solve protein folding problem and resulted in the design and successful experimental proof

of the structure of the 93-residue alfa/beta Top7 [6]. However, many improvements are

needed along these lines, and de novo generation of a functional protein is a major

challange.

To respond to this challenge, a collaborative effort between the research groups of

Baker and the Houk has led to the development of an ‘inside-out’ protocol for enzyme

design. A theoretical active site with the appropiate functionality.

Quantum Mechanical (QM) calculations are emplyod to determine the catalytic units

that will be most effective at stabilizng the transition state (TS) is determined using

Quantum Mechanical (QM) calculations. This QM optimized arrangement of a catalytic

group is known as ‘theozyme’, short for theoretical enzyme.

QM transition-state geometry is then grafted. Next, aminoacid residues surrounding

the QM theozyme are mutated and optimized to ensure good packing and fold stability and

to complement the geometric and electronic features of the TS.Selected proteins are

expressed and tested for activity for the target reaction [7].

2.2. THEOZYMES

A theozyme is a theoretical model of a catalytic site. A schematic representation of

a theozyme is given in Figure 2.2. QM calculations are carried out to generate three-

dimensional arrangements of functional groups that are optimal for stabilizing the TS of

the targeted reaction. A theozyme is typically constructed from an array of amino acid side

chains and backbone amides, but inclusion of unnatural amino acids and cofactors can

further expand the chemical space [8]. For a given reaction, a number of distinct theozyme

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motifs are usually generated, each of which varies in the composition of its functional

groups. The energy profile of each motif is computed and the magnitude of catalysis is

assessed. The theozyme motifs are further diversified geometrically by producing an

ensemble of conformations without disrupting the catalytic interactions.

Figure 2.2. Theozymes [9]

Natural protein active sites, which display a similar catalytic function, can be used as

a guide to select the initial three-dimensional arrangement of the catalytic groups for a

target reaction. The transition state geometries of these arrangements are called

‘theozymes’ [10].

2.3. INCORPORATING THEOZYMES INTO PROTEIN SCAFFOLDS : ROSETTA

MATCH

RosettaMatch is used to search the native active sites of existing protein structures

for backbone positions which can provide the three-dimensional side-chain arrangement in

a theozyme.

The program ‘’matches’’ the theozyme motif into the active site pocket by

sequentially attaching each side chain of the theozyme to the backbone of the protein

scaffold. The algorithm generates side-chain rotamers for every position in the scaffold

and pairs each rotamer with the functional groups of the theozyme [11].

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The exact three-dimensional geometry of the theozyme would be an ideal match.

However, in practice, the odds of obtaining an ideal match is vanishingly small that can

be attributed to the discrete nature of both the protein backbone and the primary

matching algorithm as well as to the computational cost associated with the mapping out

of conformational space [12].

Therefore, matching is a major bottleneck in the computational design protocol,

particuularly when a theozyme contains three or more catalytic residues. The resulting

matches are generally deviate from the theozyme geometry. For this reason, some form of

geometric filtering and ranking according to their theozyme-likeness is necessary. A

useful tool for this purpose is EDGE (enzyme design geometry evaluation). EDGE uses

geometric hashing algorithm compare theozyme atoms with a target structure and gives a

score based on the summation of their deviations.

2.3.ROSETTA-DESIGN

The RosettaDesign program is used to insert additional noncatalytic active-site

mutations that provide stabilizing interactions with the TS [13].

An initial gradient based optimization ensures that the TS is positioned such that the

catalytic inretactions are close to ideal as possible. The optimization occurs in the absence

of the other active-site residues, which have been temporarily mutated to glycine. A

reduced energy function is used that does not include solvation or attractive van der waals

terms.The optimization is followed by Monte Carlo sequence selection variations based on

the residues chosen by the user identify possible lower energy sequence . The design

phase ends within the constrained optimization of the three-dimensional structure keeping

the catalytic contacts intact [14].

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Figure 2.3. Rosetta-Match and design [15]

After the maximum number of iterations catalytic constraints are removed and side

chains are repacked and minimized. Although the process is automated, the user can input

specific mutations if required.

These steps lead to the introduction of amino acid residues that add interactions to

stabilize the position of the key catalytic residues, to optimize transition-state binding.

Each match that enters the active-site design stage contains a theozyme that is already

significantly distorted compared to the ideal QM TS geometry.

Rosetta-Design is the tasked with generating the best possible stabilization for a

geometry that in itself is non-ideal. While Rosetta-Design attempts to constrain the design

to the ideal theozyme, as specified by the geometric restraints, normally even the highest

ranked final designs differ quite considerably from the original theozyme geometry [16].

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3.METHODOLOGY

3.1.SABER MATCHING

In this project, a new approach was explored to overcome the major bottleneck,

‘matching’, in the ‘inside-out’ protocol to enzyme design.

Different from the RosettaMatch, which is based on the geometric match of the

theozyme with empty protein scaffolds. The native functional groups of natural proteins

were screened for the fundamental catalytic elements of the target reaction (Morita-Baylis-

Hillman Reaction).

SABER is an alternative to previously described matching procedure

(RosettaMatch), in which theozymes are placed into predefined active sites in selected

protein scaffolds. SABER searches the Protein Data Bank for proteins with the appropiate

catalytic functionally already in place [17]. The SABER workflow consists of the following

steps:

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Optional arrangement of catalytic functional group were taken from the

optimized theozyme geometry.

The XYZ coordinates of heteroatoms of the catalytic side chain mimics

were used to construct catalytic atom maps.

A Tess file was created including the XYZ coordinates of each atom in the catalytic

Atom Map (CAM), the PDB atom code for each atom, the PDB residue name for each

residue.After that, this file was converted to XML format to be used in the Jess search.

The Jess XML file was manually edited to allow for more diversity in the Catalytic

Atom Map. For example, consider that the original residue in the Tess file is GLU and the

atom coordinates are of the oxygen’s. An alternative option would be to search for oxygens

in the residue ASP since their oxygens are equivalent. This alternative search criteria can

be defined by the addition of an ‘or’ operator into the XML file.

The XML file was used in the JESS search.In the Jess search, PDB database was

explored for matching residues in a given Radius.

After the geometry matching step,the outputs were analyzed using the SABER

scoring functions. These includes the geometry score, which compares the RMSD of the

hit to active site model.(Theozyme).

The ActiveSiteScore, which compares the residues in the hit to the annotations in

the Catalytic Site Atom (CSA). A high score indicates that the residues in the hit

are flagged as part of an active site in the CSA.

The BuildingSiteFinder, which analyzes every hit and looks for non-water PDB

heteroatoms within 5A of the residues identified in the search. This represents

another way of identifying active/binding sites, particularly those that have not

been annotated in the CSA.

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An ideal candidate starts with a high level of geometric fidelity to the CAM,

indicated by a low GeometryScore. A hit with a high ActiveSiteScore indicates that the

residues are likely part of an existing active site [17].

4.RESULTS AND DISCUSSIONS

The Morita-Baylis-Hillman (MBH) reaction is an important atom-economical C-C

coupling reaction converting simple starting materials into highly functionalized products.

There are no natural enzymes or catalytic antibodies that can catalyze the MBH reaction.

Proteins which were designed using the inside-out approach catalyzed the MBH reaction,

but displayed rather low activities to be synthetically useful. This project aims to screen

natural proteins for potential MBH activity using state of the art computational methods

and to predict the proteins with promiscuous activity.

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Figure 4.1. Theozyme models used for Saber mathcing

The screening was carried out using SABER based on theozymes shown in Figure

4.1. The first theozyme involves a Ser-His-Asp catalytic triad mimic as a nucleophile.The

catalytic triad of Ser-His-Asp is a well known biological motif of esterases, and proteases

and lipases.It is postulated that serine hydroxyl group is activated as a nucleophile by a

proton shuttle mechanism, in which hisdine acts a general base in the deprotonation of

serine and aspartate or glutamate neutralizes the generated charge being formed on the

imidazole of histidine. Two catalytic atom maps for this theozyme were constructed from

the transition states of the conjugate addition and proton transfer steps.

The second theozyme involves an additional diglycine-back bone motif to mimic the

oxyanion hole, another well known catalytic functionality in enzymology. Oxyanion holes

in estetases, proteanes and lipases stabilize the negative charge develop on the substrate

upon nucleophilic addition with H-bond interactions. Two catalytic atom maps fort his

theozyme were constructed each of which include one of the H-bond donor groups in the

oxyanion.

4.1. THEOZYME 1 - CAM FROM CONJUGATE ADDITIONS

Figure 4.2 shows the optimized conjugate addition transition statefor theozyme 1

using density functional theory. Hetero-atoms of the Ser-His-Asp mimics were selected to

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be included in the catalytic atom map for this theozyme. XYZ coordinates of four atoms

are used to construct the Tess file.Oxygen (OG) of serine mimic , nitrogens (ND1, NE2)

of histidine mimic and the interacting oxygen of (OD1) OF aspartate mimic. These atoms

are highlighted in the figure. The tess file for this theozyme is given in Figure 4.3.

Figure 4.2. Catalytic atoms selected (highlighted) from theoyzme model THEO1

ATOM 0 OD1 ASP A 1 5.358 0.487 -0.054ATOM 0 ND1 HIS A 2 2.729 0.740 -0.089ATOM 0 NE2 HIS A 2 0.640 -0.005 -0.144ATOM 0 OG SER A 3 -1.476 -1.517 -0.306

END

Figure 4.3. The Catalytic Atom Map of Theozyme Model (Tess File) Generated for THEO1

Tess file is converted to XML files and manually modified to allow diversity.All

oxygens of carboxylate side –chain containing amino acids (Asp and Glu) were included in

the search. Instead of Serine residue only, all amino acids involving an alcohol

functionality in their side-chains (Ser, Thr,Tyr) were considered. The XML file is shown in

Figure 4.4.

<?xml version="1.0"?><jess xmlns="http://www.ebi.ac.uk/~jbarker/Jess"> <template name="cam-tm6-ts12.tess"> <atom name="0"> <select> and ( or

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( and ( or ( isAtomNamed("_OD2"), isAtomNamed("_OD1") ), inResidueNamed("ASP") ), and ( or ( isAtomNamed("_OE2"), isAtomNamed("_OE1") ), inResidueNamed("GLU") ) ) ) </select> <xyz>5.359 0.487 -0.054</xyz> </atom> <atom name="1"> <select> and ( or ( isAtomNamed("_NE2"), isAtomNamed("_ND1") ), inResidueNamed("HIS"), inAnnulus(atom(#0),sub(2.64,$delta),add(2.64,$delta)), inChainOf(atom(#0)) ) </select> <xyz>2.730 0.740 -0.089</xyz> </atom> <atom name="2"> <select> and ( or ( isAtomNamed("_ND1"), isAtomNamed("_NE2") ), inResidueNamed("HIS"), inAnnulus(atom(#0),sub(4.74,$delta),add(4.74,$delta)), inChainOf(atom(#0)), inAnnulus(atom(#1),sub(2.22,$delta),add(2.22,$delta)) ) </select> <xyz>0.641 -0.006 -0.145</xyz> </atom> <atom name="3"> <select> and ( or ( and ( isAtomNamed("_OG_"), inResidueNamed("SER")

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), and ( isAtomNamed("_OG1"), inResidueNamed("THR") ), and ( isAtomNamed("_OH_"), inResidueNamed("TYR") ) ), inAnnulus(atom(#0),sub(7.13,$delta),add(7.13,$delta)), inChainOf(atom(#0)), inAnnulus(atom(#1),sub(4.78,$delta),add(4.78,$delta)), inAnnulus(atom(#2),sub(2.61,$delta),add(2.61,$delta)) ) </select> <xyz>-1.476 -1.517 -0.306</xyz> </atom> </template>

</jess>

Figure 4.4. The Catalytic Atom Map in Jess XML format

The XML file ( Figure 4.4) describing the Catalytic Atom Map was run on SABER

to find matches to CAM in the PDB database and to analyze the matching hits. The PDB

IDs of seven hits proteins with lowest root mean square deviation (RMSD) from the

quantum mechanically optimized arrangement of catalytiic atoms are listed in Table X

along with their corresponding RMSD values and matching amino acids.

Table 4.1. Matches for THEO1

PDB ID Name of Protein RMSD

(Å)Matching Residue

1I8D Riboflavin synthase 0.76

ASP B 185HIS B 97

SER B 146

1HZW Thymidylate synthase 1.11

ASP A 218HIS A 256SER A 216

1AXW Thymidylate synthase 1.25

ASP B 169HIS B 207SER B 167

1HW3 Thymidylate synthase

1.2ASP A 218HIS A 256SER A 216

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1HVY Thymidylate synthase 1.18

ASP B 218HIS B 256SER B 216

1F28 Thymidylate synthase 1.24

ASP D 202HIS D 240SER D 200

1CHD Cheb methylesterase 1.08

ASP A 286HIS A 190SER A 164

The matching residues in these protein structures were pair-fitted with the theozyme

model used to construct the CAM for this search using PYmol. Five out of seven of the hits

are thymidylate syntheses…

One of the seven of the hits is an esterase, for which the Ser-His-Asp catalytic triad

plays a major role in this catalytic activity.

The lowest RMSD was found as 0.76 Å for 18ID.

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Figure 4.5. Pymol Overlay of the watching protein active site (18ID) and the theozyme

model THEO1

Figure 4.5 represents the matching with 18ID and THEO1. Green lines are theozyme,

blue lines represents native proteins. Although the pair-fitting resulted in no steric clashes

of the substrate in the active site, the matching Asp group interacts with …..

Rather than the His residue, which way severely disrupt the proton shuttle mechanism

hence unfavorably affect the nucleophilicity of Serine.

4.2.THEOZYME 2- CAM FROM PROTON TRANSFER TS

Figure 4.6 shows the proton transfer transition state for theozyme 2 using density

functional theory. Ser-His-Asp mimics were selected for the catalytic atom map for this

theozyme. XYZ coordinates of these atoms are used to construct the Tess file. Oxygen

(OG) of serine mimic, nitrogens (ND1, NE2) of histidine mimic and the interacting

oxygen of (OD1) of aspartate mimic. These four atoms are highlighted in the figure

which is below. The tess file for this theozyme is given in Figure 4.5.

Figure 4.6. Catalytic atoms selected (highlighted) from theozyme model Proton Transfer

TS

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ATOM 0 OD1 ASP A 1 -6.504 -1.210 -0.106ATOM 0 ND1 HIS A 2 -4.434 0.494 -0.257ATOM 0 NE2 HIS A 2 -2.308 1.032 0.004ATOM 0 OG SER A 3 0.238 1.853 0.650END


Tess file is converted to XML files and manually modified to allow diversity. All



functionality in their side-chains (Ser, Thr, Tyr) were considered. The XML file is shown

in Figure 4.7.

<?xml version="1.0"?><jess xmlns="http://www.ebi.ac.uk/~jbarker/Jess"> <template name="cam-tm6-ts3.tess"> <atom name="0"> <select> and ( or ( and ( or ( isAtomNamed("_OD2"), isAtomNamed("_OD1") ), inResidueNamed("ASP") ), and ( or ( isAtomNamed("_OE2"), isAtomNamed("_OE1") ), inResidueNamed("GLU") ) ) ) </select> <xyz>-6.504 -1.210 -0.107</xyz> </atom> <atom name="1"> <select> and ( or ( isAtomNamed("_NE2"), isAtomNamed("_ND1") ),

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inResidueNamed("HIS"), inAnnulus(atom(#0),sub(2.68,$delta),add(2.68,$delta)), inChainOf(atom(#0)) ) </select> <xyz>-4.435 0.494 -0.258</xyz> </atom> <atom name="2"> <select> and ( or ( isAtomNamed("_NE2"), isAtomNamed("_ND1") ), inResidueNamed("HIS"), inAnnulus(atom(#0),sub(4.76,$delta),add(4.76,$delta)), inChainOf(atom(#0)), inAnnulus(atom(#1),sub(2.21,$delta),add(2.21,$delta)) ) </select> <xyz>-2.308 1.033 0.004</xyz> </atom> <atom name="3"> <select> and ( or ( and ( isAtomNamed("_OG_"), inResidueNamed("SER") ), and ( isAtomNamed("_OG1_"), inResidueNamed("THR") ), and ( isAtomNamed("_OH_"), inResidueNamed("TYR") ), ), inAnnulus(atom(#0),sub(7.44,$delta),add(7.44,$delta)), inChainOf(atom(#0)), inAnnulus(atom(#1),sub(4.95,$delta),add(4.95,$delta)), inAnnulus(atom(#2),sub(2.75,$delta),add(2.75,$delta)) ) </select> <xyz>0.239 1.853 0.650</xyz> </atom> </template></jess>



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PDB ID Name of Protein RMSD (Å) Matching Residue

1HT3 Proteinase 1.53ASP A 39HIS A 69

SER A 224

1A88 Chloroperoxidase 1.45ASP A 226HIS A 255SER A 96

1BS9 Acetyl xylan esterase 1.47ASP A 175HIS A 187SER A 90

1CUS Cutinase 1.49 ASP A 175HIS A 188SER A 120

1FJ2 Acyl protein thioesterase 1.49ASP B 169HIS B 203SER B 114


model used to construct the CAM for this seacrh using PYmol. Five out of seven of the hits

are thymidylate syntheses……



The lowest RMSD was found for 1BS9 (Acetyl Xylan Esterase).The RMSD value of

this protein is 1.47 Å.

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Figure 4.9. Pymol Overlay of the watching protein active site (1BS9) and the theozyme

model THEO2

Figure 4.9 represents the matching with 1BS9 and THEO2. Green lines are

theozyme, blue lines represents native proteins. Although the pair-fitting resulted in no

steric clashes of the substrate in the active site, the matching Asp group interacts with …..



4.3. THEOZYME 3




are used to construct the Tess file.Oxygen (OG) of serine mimic, nitrogens (ND1, NE2) of

histidine mimic and the interacting oxygen of (OD1) OF aspartate mimic. These atoms


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Figure 4.10. Catalytic atoms selected(highlighted) from theoyzme model THEO3

ATOM 0 OD1 ASP A 1 7.607 0.909 0.085ATOM 0 ND1 HIS A 2 4.982 0.936 0.023ATOM 0 NE2 HIS A 2 2.949 0.025 -0.102ATOM 0 OG SER A 3 1.148 -1.694 -0.292ATOM 0 ND2 ASN A 4 -3.461 1.749 -0.816END

Figure 4.11. The Catalytic Atom Map of Theozyme Model (Tess File) Generated for

THEO3





in Figure 4.12.

<?xml version="1.0"?><jess xmlns="http://www.ebi.ac.uk/~jbarker/Jess"> <template name="cam-ts-tox6-ts12-n1_1.tess"> <atom name="0"> <select> and ( or ( and ( or

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( isAtomNamed("_OD2"), isAtomNamed("_OD1") ), inResidueNamed("ASP") ), and ( or ( isAtomNamed("_OE2"), isAtomNamed("_OE1") ), inResidueNamed("GLU") ) ) ) </select> <xyz>7.607 0.910 0.085</xyz> </atom> <atom name="1"> <select> and ( or ( isAtomNamed("_NE2"), isAtomNamed("_ND1") ), inResidueNamed("HIS"), inAnnulus(atom(#0),sub(2.62,$delta),add(2.62,$delta)), inChainOf(atom(#0)) ) </select> <xyz>4.983 0.937 0.024</xyz> </atom> <atom name="2"> <select> and ( or ( isAtomNamed("_NE2"), isAtomNamed("_ND1") ), inResidueNamed("HIS"), inAnnulus(atom(#0),sub(4.74,$delta),add(4.74,$delta)), inChainOf(atom(#0)), inAnnulus(atom(#1),sub(2.23,$delta),add(2.23,$delta)) ) </select> <xyz>2.949 0.026 -0.103</xyz> </atom> <atom name="3"> <select> and ( or ( and ( isAtomNamed("_OG_"), inResidueNamed("SER") ), and

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( isAtomNamed("_OG1"), inResidueNamed("THR") ), and ( isAtomNamed("_OH_"), inResidueNamed("TYR") ) ), inAnnulus(atom(#0),sub(6.97,$delta),add(6.97,$delta)), inChainOf(atom(#0)), inAnnulus(atom(#1),sub(4.66,$delta),add(4.66,$delta)), inAnnulus(atom(#2),sub(2.50,$delta),add(2.50,$delta)) ) </select> <xyz>1.149 -1.694 -0.293</xyz> </atom> <atom name="4"> <select> and ( or ( and ( isAtomNamed("_N__"), inResidueNamed("GLY") ), and ( isAtomNamed("_N__"), inResidueNamed("ALA") ), and ( isAtomNamed("_ND2"), inResidueNamed("ASN") ), and ( isAtomNamed("_NE2"), inResidueNamed("GLN") ) ), inAnnulus(atom(#0),sub(11.14,$delta),add(11.14,$delta)), inChainOf(atom(#0)), inAnnulus(atom(#1),sub(8.53,$delta),add(8.53,$delta)), inAnnulus(atom(#2),sub(6.68,$delta),add(6.68,$delta)), inAnnulus(atom(#3),sub(5.78,$delta),add(5.78,$delta)) ) </select> <xyz>-3.462 1.750 -0.816</xyz> </atom> </template></jess>



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PDB ID Name of Protein RMSD (Å)

Matching Residue

1S0R Trypsinogen 0.41

ASPA84HISA40

SERA177 GLNA174

1R1D Carboxylesterase 0.43

ASPB192 HISB222SERB93GLYB26

3HJR Extracellular serine protease 0.44

ASPA78HISA115SERA336 ASNA333

1AU9 Subtisilin 0.45

ASPA32 HISA64SERA221 GLYA219


model used to construct the CAM fort his seacrh using PYmol. Five out of seven of the hits

are thymidylate syntheses……



The lowest RMSD was found for……

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Figure 4.13. Pymol Overlay of the watching protein active site (1R1D) and the theozyme

model THEO3

The matching between 1R1D and THEO3 is shown in Figure 4.13. Green lines are

theozyme, blue lines represnts native proteins. Although the pair-fitting resulted in no




4.4. THEOZYME 4




are used to construct the Tess file.Oxygen (OG) of serine mimic , nitrogens (ND1, NE2)

of histidine mimic and the interacting oxygen of (OD1) OF aspartate mimic. These atoms


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Figure 4.14. Catalytic atoms selected(highlighted) from theoyzme model THEO4

ATOM 0 OD1 ASP 7.607 0.909 0.085ATOM 0 ND1 HIS 4.982 0.936 0.023ATOM 0 NE2 HIS 2.949 0.025 -0.102ATOM 0 OG SER 1.148 -1.694 -0.292ATOM 0 N GLY -5.654 0.672 0.624END






in Figure 4.15.

<?xml version="1.0"?><jess xmlns="http://www.ebi.ac.uk/~jbarker/Jess"> <template name="cam-ts-tox6-ts12-n2.tess"> <atom name="0"> <select> and ( or (

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and ( or ( isAtomNamed("_OD2"), isAtomNamed("_OD1") ), inResidueNamed("ASP") ), and ( or ( isAtomNamed("_OE2"), isAtomNamed("_OE1") ), inResidueNamed("GLU") ) )

) </select> <xyz>7.607 0.910 0.085</xyz> </atom> <atom name="1"> <select> and ( or ( isAtomNamed("_NE2"), isAtomNamed("_ND1") ), inResidueNamed("HIS"), inAnnulus(atom(#0),sub(2.62,$delta),add(2.62,$delta)), inChainOf(atom(#0)) ) </select> <xyz>4.983 0.937 0.024</xyz> </atom> <atom name="2"> <select> and ( or ( isAtomNamed("_NE2"), isAtomNamed("_ND1") ), inResidueNamed("HIS"), inAnnulus(atom(#0),sub(4.74,$delta),add(4.74,$delta)), inChainOf(atom(#0)), inAnnulus(atom(#1),sub(2.23,$delta),add(2.23,$delta)) ) </select> <xyz>2.949 0.026 -0.103</xyz> </atom> <atom name="3"> <select> and ( or ( and ( isAtomNamed("_OG_"), inResidueNamed("SER")

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), and ( isAtomNamed("_OG1"), inResidueNamed("THR") ), and ( isAtomNamed("_OH_"), inResidueNamed("TYR") ) ), inAnnulus(atom(#0),sub(6.97,$delta),add(6.97,$delta)), inChainOf(atom(#0)), inAnnulus(atom(#1),sub(4.66,$delta),add(4.66,$delta)), inAnnulus(atom(#2),sub(2.50,$delta),add(2.50,$delta)) ) </select> <xyz>1.149 -1.694 -0.293</xyz> </atom> <atom name="4"> <select> and ( or ( and ( isAtomNamed("_N__"), inResidueNamed("GLY") ), and ( isAtomNamed("_N__"), inResidueNamed("ALA") ), and ( isAtomNamed("_ND2"), inResidueNamed("ASN") ), and ( isAtomNamed("_NE2"), inResidueNamed("GLN") ) ), inAnnulus(atom(#0),sub(11.14,$delta),add(11.14,$delta)), inChainOf(atom(#0)), inAnnulus(atom(#1),sub(8.53,$delta),add(8.53,$delta)), inAnnulus(atom(#2),sub(6.68,$delta),add(6.68,$delta)), inAnnulus(atom(#3),sub(5.78,$delta),add(5.78,$delta)) ) </select> <xyz>-3.462 1.750 -0.816</xyz> </atom> <atom name="5"> <select> and ( or ( and ( isAtomNamed("_N__"), inResidueNamed("GLY") ),

28

and ( isAtomNamed("_N__"), inResidueNamed("ALA") ), and ( isAtomNamed("_ND2"), inResidueNamed("ASN") ), and ( isAtomNamed("_NE2"), inResidueNamed("GLN") ) ), inAnnulus(atom(#0),sub(13.27,$delta),add(13.27,$delta)), inChainOf(atom(#0)), inAnnulus(atom(#1),sub(10.66,$delta),add(10.66,$delta)), inAnnulus(atom(#2),sub(8.66,$delta),add(8.66,$delta)), inAnnulus(atom(#3),sub(7.26,$delta),add(7.26,$delta)), inAnnulus(atom(#4),sub(2.84,$delta),add(2.84,$delta)) ) </select> <xyz>-5.654 0.673 0.625</xyz> </atom> </template></jess>



PDB ID Name of Protein RMSD (Å) Matching Residue

3DYV Esterase 0.32

ASP A 188HIS A 219SER A 94GLY A 26

2ES4 Lipase 0.34

ASP A 263HIS A 285SER A 87ALA A 18

29

1CVL Triacylglycerol hydrolase 0.34

ASP A 263HIS A 285SER A 87ALA A 18

1X12 Pyrrolidone-carboxylate peptidase 0.36

GLU A 79HIS A 166SER A 142ASN A 17


model used to construct the CAM for this seacrh using PYmol. Five out of seven of the

hits are thymidylate syntheses……



The lowest RMSD was found as 0.32 Å for 3DYV.

30

Figure 4.17. Pymol Overlay of the watching protein active site (3DYV) and the theozyme

model THEO2

The matching between 3DYV and THEO4 is shown in Figure 4.17. Green lines are

theozyme, blue lines represnts native proteins. Although the pair-fitting resulted in no




5.CONCLUSION AND RECOMMENDATION

5.1.CONCLUSION

Chemical catalysts have lower the activation barier so they are very important.Due to

environmental issues, looking for a way of enzyme usage instead of chemical catalysts has

started. Usually, the way used is to modify a natural enzyme through protein engineering

with respect to the desired application.

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The aim of the project was to determine natural promising proteins is for MBH activity

using computational methods and to predict the proteins activity and redesign the most

promising proteins.

Theoretical enzyme active site models was purposed to generate using various amino acid

mimics.

32

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