Jeff Hamilton
Protein Modeling of the Heterocompound Flavin
Adenine Dinulecotide and of the Protein VanillylAlcohol Oxidase
Introduction
The dimeric protein Vanillyl-Alcohol Oxidase from Penicillium simplicissimum
catalyzes the oxidation of some 4-hydroxybenzyl alcohols. For example, VanillylAlcohol Oxidase catalyzes the oxidation of vanillyl alcohol to produce canillin and
hydrogen peroxide. It has the heterocompound Flavin adenine dinucleotide attached to
the protein and it aids in catalyzing the reaction.
Protein: Vanillyl-Alcohol Oxidase
Protein number: 1AHZ
Heterocompound: Flavin Adenine Dinucleotide
Het code: FAD
Part I
The heterocompound protein Flavin Adenine Dinucleotide and the VanillylAlcohol Oxidase which complexes with FAD was selected. The file of the complex was
downloaded from the RCSB website to the program DS Visualizer. The heterocompound
was extracted from the complex and the geometry for FAD was corrected.
Figure 1: Heterocompound FAD
Part II
In DS Visualizer, the protein was shown as a solid ribbon format. The
heterocompound is within the protein and it is displayed in a ball and stick format with
the different elements displayed in different colors.
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Jeff Hamilton
Figure 2: The protein in solid ribbon form with the Heteroatom
Part III
The PDB file of the heterocompound was imported into Chem3D Ultra. The
steric energy of the heterocompound was calculated. Then the program was used to
perform a energy minization of the heteroatom (Figure 4).
Table 1: Steric Energy and Energy Minimization Calculations of the FAD
Energy Terms Steric Energy Energy Minimization
Stretch:
100.7771
174.1773
Bend:
4365.8746
525.8078
Stretch-Bend:
-12.389
5.6692
Torsion:
9.3069
98.4615
Non-1,4 VDW:
49.096
3.3126
1,4 VDW:
35.7321
50.1307
Charge/Charge:
0
0
Charge/Dipole:
17.4266
-14.6333
Dipole/Dipole:
0
-18.7167
Total:
4565.8243
824.2092
Figure 3: FAD before Minimization
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Jeff Hamilton
Figure 4: FAD after Minimization
The steric energy of Flavin Adenine Dinucleotide (FAD) was calculated. When
FAD’s energy was minimized, it represents the energy minimum of the compound in the
gas phase.
Stretch is the energy related to energy of the bonds due to the stretching of the
bonds of the protein. The steric energy before the minimization was about 74 kcal/mol
lower than the energy after minimization. This is due to the bending of FAD around the
P-O bonds of the phosphate groups. Bend is the energy related to the bond angles that
keep the bonds from the most stable angle or conformation. The Bend for the gas phase
heterocompound was about 3840 kcal/mol less then the bend for the heterocompound
before minimization. This is due to the high van der Waals forces between electron
clouds in the protein before minimization which causes the bend energy to be high. The
bend contributed the most energy to the steric energy of both compounds. The stretchbend is the energy that involves two bonds that form a bond angle and when that bond is
strained. The heterocompound has about 17 kcal/mol less stretch-bend energy then the
heterocompound in the gas phase. This is due to the bending of the energy minimized
oxidase around the P-O bonds. The stretch bend contributed little to the overall steric
energy of both compounds.
The torsion is the energy from deviating dihedral angles from their most favorable
values. The torsion energy for the heterocompound was about 88 kcal/mol less then the
heterocompound in the gas phase. This shows that the heterocompound before
minimization has more stability from angles that are closer to their optimal dihedral angle.
The non-1,4 VDW is the energy related to the repulsive forces of the electron clouds
between atoms that are farther then three atoms apart. The FAD in the gas phase had
about 46 kcal/mol less non-1,4 VDW steric energy then the FAD from the heterocomplexed compound. This is due to the bending of FAD in the gas phase to produce
more favorable interactions. The 1,4 VDW is the energy from the repulsion of the
electron clouds of the atoms that are two atoms apart. The 1,4 VDW of the FAD in the
gas phase was about 15 kcal/mol higher then the FAD from the hetero-complexed protein
since the bending caused the oxygens around the phosphate groups to be closer together,
causing more repulsions as the molecule bend to its energy minimized conformation.
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Jeff Hamilton
The charge/charge is the sum of the pairs of electrostatic interactions between
charged atoms. The charge/charge interactions in both of the phases of the FAD turned
out to be zero. The electrostatic interactions between the two compounds did not change
from the minimization. The charge/dipole is the electrostatic energy from the interaction
of a charged group and of a group with a dipole. The charge/dipole of the FAD in the gas
phase is about 31 kcal/mol lower then the FAD before minimization. The FAD in the gas
phase had more favorable dipole/charge interactions then the FAD from the heterocomplexed compound. Dipole/dipole is the energy of the interactions between two
groups with dipoles. The energy minimized compound had a dipole/dipole that was 18
kcal/mol lower since it was in a conformation that allowed more dipole interactions
between the lone pairs of the oxygens and the hydrogens bonded to carbon. The total
energy is the sum of all the steric energies of the compound. The FAD in the gas phase is
much more stable then the FAD from the hetero-complexed oxidase since the FAD in the
gas phase had about 5740 kcal/mol less of steric energy within the compound.
Part IV
On Chem3D Ultra, an overlay of the energy minimized heterocompound and the
heterocompound from the hetero-complexed protein was performed.
Figure 5: Overlay of the Two FAD compounds
Part V
The amino acid sequence of the protein was obtained from the wiring diagram in
the PDBsum site (Figure 6). The red dots indicate all of the amino acids of the protein
that interact with the heterocompound and the amino acid. The amino acids in red boxes
are in the active site of the protein. The ligplot of the protein was obtained from the
PDBsum site and it shows the amino acids of the protein that interact with the
heterocompound (Figure 7). The amino acids that interact with FAD where highlighted
in yellow in DS Visualizer.
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Jeff Hamilton
Figure 6: Wiring Diagram of the Protein
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Jeff Hamilton
Figure 7: Ligplot of the
Heterocompound FAD
Figure 8: Amino acids that interact with
FAD in DS Visualizer (amino acids that
did not interact with FAD where not
shown)
The LIGPLOT shows the interactions between the amino actions of the protein such as
the hydrogen bonds (dotted green lines). The nitrogen of Arg 504 hydrogen bonds with
the lone electron pairs of the oxygen which is a part of a phosphate group.
The “eyelashes” are the hydrophobic interactions or other non-hydrogen bonding
interactions that occur between the amino acids and FAD. For example, the hydrophobic
amino acid Trp 413 interacts with the non-polar methylene group of FAD. Also the
oxygen of Glu 260 interacts with the lone pairs of electrons on the nitrogen of the adenine
of the FAD molecule.
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Jeff Hamilton
Figure 9: Flavin adenine Dinucleotide (ball and stick) within the Vanillyl-Alcohol
Oxidase (solid ribbon)
Figure 10: The amino acids that interact with FAD are shown in a pale yellow
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Jeff Hamilton
Figure 11: The hetero-complex compound showing the interactions between FAD and
the amino acid side chains.
Part VI
The amino acids that interact with FAD were identified and analyzed in Table 2.
Table 2: Amino Acids that interact with FAD
Amino acid
Hetero
residue
compound
atoms
Nature of interaction
Val 262
Adenine
nitrogens
The C-O and the N-H of the Val are hydrogen
bonding with the N-H and N respectively of the
heterocompound.
Lys 545
Ribose hydroxyl
The NH of Lys 545 is hydrogen bonding to the
two hydroxyls of the ribose in the adenylate
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Ser 101
Ile 102
Adenine
nitrogen and
phosphate group
ribose
The O-H and the N-H of Ser in hydrogen bonding
to the nitrogen and the oxygen of the phosphate
respectively.
The –CH3 of Ile is hydrophobically interacting
with the –CH2 of the ribose.
Gly 103
Phosphate group
The N-H of Gly is hydrogen is bonding to the
oxygen of the phosphate in FAD
Asn 105
Phosphate group
The N-H of the amide group of Asn is hydrogen
bonding to the oxygen of the phosphate of FAD.
Glu 182
“
“
Asn 179
Amide group of
the fused
aromatic rings
Arg 504
Amide group of
the fused
aromatic rings
The amide group of Asn 179 is hydrogen bonding
to the C=O of the amide group of FAD and to the
hydroxyl group that is in between the nucleotide
and the fused aromatic rings.
The amide group of Asn 179 is hydrogen bonding
to the C=O of the amide group of FAD and to the
hydroxyl group that is in between the nucleotide
and the fused aromatic rings.
Val 185
The nitrogen in
the fused
aromatic rings
Fused Aromatic
rings
“
Pro 169
Gly 184
The C=O of Val in the peptide bond is hydrogen
bonding to the nitrogen in the fused aromatic rings
Hydrophobic interactions between the praline
methyl group and the fused aromatic rings
“
Tyr 187
C=O of the fused The hydroxyl group of the tyrosine is hydrogen
aromatic rings
bonding to the carbonyl of the fused aromatic
rings
Asp 170
C=O of the fused The N-H of the amide group of Asp is hydrogen
aromatic rings
bonding to the carbonyl of the fused aromatic
rings
Trp 413
aromatic ring
The aromatic Trp 413 is hydrophobically
interacting with one of the fused aromatic rings.
Arg 104
Phosphate group
The N-H and the C=O of the peptide bond of Arg
are hydrogen bonding to the oxygen of the
phosphate group and the hydroxyl group that is in
between the nucleotide and the fused aromatic
rings.
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Jeff Hamilton
Ser 175
Phosphate group
Phe 424
Fused aromatic
rings
The N-H of the peptide bond of Ser is hydrogen
bonding to the oxygen of the phosphate group
The aromatic Phe 424 is hydrophobically
interacting with one of the fused aromatic rings.
Gly 184
Pro 169
“
Fused aromatic
rings
“
The methyl of Pro is hydrophobically interacting
with one of the fused aromatic rings.
Glu 182
“
“
Part VII
Bibliography:
1. Andrea Mattevi, Marco W. Fraaije, Alessandro Coda, Willem J.H. van Berkel
Crystallization and preliminary x-ray analysis of the flavoenzyme vanillyl-alcohol
oxidase from Penicillium Simplicissimum. (4):601-3. 1997 Apr. 27.
http://www3.interscience.wiley.com/cgi-bin/fulltext/52413/PDFSTART
2. H.M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T.N. Bhat, H. Weissig, I.N.
Shindyalov, P.E. Bourne: The Protein Data Bank. Nucleic Acids Research, 28 pp.
235-242 (2000).
3. Laskowski R A, Chistyakov V V, Thornton J M (2005). PDBsum more: new
summaries and analyses of the known 3D structures of proteins and nucleic acids.
Nucleic Acids Res., 33, D266-D268. http://www.ebi.ac.uk/pdbsum/
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