Modeling the E. coli 4-hydroxybenzoic acid oligoprenyltransferase (ubiA transferase) and characterization of potential active sites
- First Online:
- Cite this article as:
- Bräuer, L., Brandt, W. & Wessjohann, L.A. J Mol Model (2004) 10: 317. doi:10.1007/s00894-004-0197-6
- 202 Views
4-Hydroxybenzoate oligoprenyltransferase of E. coli, encoded in the gene ubiA, is an important key enzyme in the biosynthetic pathway to ubiquinone. It catalyzes the prenylation of 4-hydroxybenzoic acid in position 3 using an oligoprenyl diphosphate as a second substrate. Up to now, no X-ray structure of this oligoprenyltransferase or any structurally related enzyme is known. Knowledge of the tertiary structure and possible active sites is, however, essential for understanding the catalysis mechanism and the substrate specificity.
With homology modeling techniques, secondary structure prediction tools, molecular dynamics simulations, and energy optimizations, a model with two putative active sites could be created and refined. One active site selected to be the most likely one for the docking of oligoprenyl diphosphate and 4-hydroxybenzoic acid is located near the N-terminus of the enzyme. It is widely accepted that residues forming an active site are usually evolutionary conserved within a family of enzymes. Multiple alignments of a multitude of related proteins clearly showed 100% conservation of the amino acid residues that form the first putative active site and therefore strongly support this hypothesis. However, an additional highly conserved region in the amino acid sequence of the ubiA enzyme could be detected, which also can be considered a putative (or rudimentary) active site. This site is characterized by a high sequence similarity to the aforementioned site and may give some hints regarding the evolutionary origin of the ubiA enzyme.
Semiempirical quantum mechanical PM3 calculations have been performed to investigate the thermodynamics and kinetics of the catalysis mechanism. These results suggest a near SN1 mechanism for the cleavage of the diphosphate ion from the isoprenyl unit. The 4-hydroxybenzoic acid interestingly appears not to be activated as benzoate anion but rather as phenolate anion to allow attack of the isoprenyl cation to the phenolate, which appeared to be the rate limiting step of the whole process according to our quantum chemical calculations. Our models are a basis for developing inhibitors of this enzyme, which is crucial for bacterial aerobic metabolism.
Figure Structure of the model of ubiA oligoprenyltransferase derived from the photosynthetic reaction center (1PRC). Putative active amino acid residues and substrates are shown as capped sticks to describe their location and geometry in the putative active sites. The violet spheres identify Mg2+.
KeywordsTransferasesPrenylationHomology modelingBiocatalysis mechanism
3D protein secondary structure prediction
4-hydroxybenzoic acid (para-hydroxybenzoat)
basic local alignment search tool
blocks substitution matrix
genetic optimization ligand docking
human immunodeficiency virus
molecular operating environment
percent accepted mutation
protein data bank
parametrized method 3
- PP or DPP
protein structure analysis
tripos associated force field
The enzyme was discovered 1972 by Young et al.  in E. coli cell extracts. The corresponding gene was located on the physical map of E. coli by Nishimura et al.  and cloned by Heide et al.  and Nichols et al.  It consists of 290 amino acids and is membrane bound. Solubilization of the enzyme in detergents leads to fast and usually irreversible loss of activity. Furthermore, magnesium ions are essential for the catalytic activity. No X-ray structure of a 4-hydroxybenzoate oligoprenyltransferase or any homologous protein is known. Aromatic prenyltransferases are still largely white spots for structural biology. In order to understand the reaction mechanism and to support the design of selective inhibitors, knowledge of the tertiary structure and the active site is essential since 3-oligoprenyl-4-hydroxybenzoic acid is a direct precursor of ubiquinones (coenzymes Qn). The specific inhibition of the biosynthesis of this compound should disrupt cellular respiration and thus affect bacterial growth. Additionally, the enzyme has been used successfully as a biocatalyst to form different C–C bonds under mild reaction conditions.  Modeling may also aid the design of alternative clones with a different, non-natural substrate spectrum.
Here we will describe a first 3D structural model of a member of this family of membrane bound prenyltransferases, including the characterization of two putative active sites and studies of the enzymatic catalysis mechanism.
Materials and methods
All calculations were performed on Silicon Graphics Octane workstations and personal computers. Structures were graphically displayed, modified and evaluated using SYBYL and STEREOGRAPHIC stereoglasses.  MOE, a molecular modeling program package, was used to prepare different models of 4-hydroxybenzoate oligoprenyltransferase. The homology modeling procedure inside MOE is automated. The template(s) and the substitution matrix and other parameters, e.g. gap penalties, must only be defined for an appropriate alignment between the template and target sequence. Briefly, the modeling procedure consists of the following steps: comparable enzymes with known X-ray structures from the PDB were aligned with the sequence of 4-hydroxybenzoate oligoprenyltransferase. 
For all calculations and structural refinements of the models without any ligand, the AMBER all atom force field, included in the modeling packages described above, was used.  The AMBER force field is very well suited for calculations of proteins, but due to the lack of parameters for the ligands the TRIPOS associated force field (TAFF) was used for calculations of protein–ligand complexes.  During all these simulations the backbone atoms of the protein were fixed.
Ten slightly different models were created with MOE and subsequently minimized by means of the AMBER force field including electrostatic interactions based on AMBER partial charge distributions. The final structure refinement (including lateron the ligands) of the resultant models was done using the TAFF implemented in SYBYL. The quality of the structures obtained was checked with PROSAII.  The program calculates the energy potentials for the atomic interactions of all amino acid residue pairs as a function of the distance between the corresponding atoms. The energies of all conformations that exist in an integrated data base with respect to the given sequence are calculated using the potential of mean force derived by statistical analysis of a set of natively folded proteins. Negative energies of a PROSAII plot indicate that the modeled structures may represent a native fold, whereas sequences with positive energies must be inspected critically. Due to the membrane location of the enzyme, only the Cα and Cβ interactions are inspected, instead of including surface potentials, which would only be relevant for water-soluble proteins.
Different substrates (e.g. 4-hydroxybenzoate, geranyl diphosphate, octaprenyl diphosphate) were docked inside suggested active sites of the models. The docking of the substrates was done manually as well as automated. The program used for automated docking was GOLD (Genetic Optimized Ligand Docking), which uses genetic algorithms to find appropriate docking arrangements of ligands into receptor binding sites.  Additionally, the docking arrangements were refined using molecular dynamic simulations with subsequent energy minimization using the TAFF. To simulate an induced fit, the ligands and the side chains of the protein were considered flexible whereas all backbone atoms of the protein were fixed. Secondary structure predictions, alignments, multiple alignments, and other diagnostics were done by means of methods available on the internet. To align the sequences we used program routines implemented in SYBYL/COMPOSER. As substitution matrix we applied PMUTATION. The gap penalty was set to 8. The calculated identity score (% identity) is the number of identical residues in the two sequences divided by the length of the shortest sequence (without gaps). Semiempirical quantum mechanical calculations were carried out using the program package Spartan.  The program parameters and options for these calculations are given in detail in the relevant section of this article.
Search for homologous proteins and structural similarities
Proteins with highest similarity scores to 4-hydroxybenzoic acid oligoprenyltransferase and X-ray structural data available. The calculated overall hydrophobicity of the X-ray structures is given by log P
Name and source of the protein
Overall hydrophobicity (log P)
5-Epi-aristolochene synthase (Nicotiana tabacum)
Geranylgeranyltransferase (Rattus norvegicus)
Photosynthetic reaction centre (Rhodopseudomonas viridis)
Farnesyl pyrophosphate synthetase (Gallus gallus)
Glycerol facilitator (Escherichia coli)
Human estrogen receptor (Homo sapiens)
Old yellow enzyme (Saccharomyces pastorianus)
Dihydropteroate synthetase (Staphylococcus aureus)
Amino acid sequence alignment of the photosynthetic reaction centre protein (1prc) compared to predicted positions of secondary structural elements of the ubiA-enzyme, using phd and 3D-pssm
The bold letters designate amino acids of α-helical regions, which were predicted by both programs, phd and 3D-pssm. All other residues show loop regions, elements with indifferent secondary structure. In no case, sheet elements were predicted.
Alignment of partial sequences of 4-hydroxybenzoic acid octaprenyltransferases
Homology modeling and structure refinement
The quality and stereochemistry of the three-dimensional structure of the model was evaluated and analyzed using PROCHECK at a theoretical resolution of 2.0 Å.  The dihedral angles (Phi and Psi) are localized only in most favored and additionally allowed regions of the Ramachandran plot.
Docking studies and characterization of the active sites
As shown in Fig. 1 the active site 1 is situated in a loop region, probably outside of the cell membrane, but directly at the surface of the cell membrane. Because of the hydrophobicity of longer prenyl side chains, we expect these substrates to be located in the membrane, whereas the diphosphate sticks out and is able to dock into the active site cavity. The other presumed active site 2 is located on the opposite site, in the interior of the enzyme and within the membrane area, but perhaps still reachable from the cytosol.
At this site, a magnesium ion forms a tetrahedral metal complex with the two oxygen atoms of Asp75 and two oxygens of the diphosphate substrate. The theoretically remaining two ligand sites to form an octahedral ligand sphere at the metal are not occupied. The binding of the magnesium ion is very similar to the arrangement found in the active site of farnesyl diphosphate synthetase (pdb code: 1UBY), where a magnesium ion is simultaneously complexed by an aspartate and a dimethylallyl diphosphate. Furthermore, in our model 4-hydroxybenzoate forms a salt bridge of its carboxyl group to the side chain of Arg137 and the phenolic hydroxy group of the benzoic acid derivative forms a hydrogen bond to Asp71. In addition to the binding of the diphosphate moiety via complexation of the magnesium ion, the docking arrangement is stabilized by hydrophobic interactions of the organic oligoprenyl chain, especially with Leu141 and Leu256 of the enzyme. In this way, the two substrates are bound close to each other in the active site. In order to test the relevance of the model, (2-Z)-octaprenyl diphosphate was also docked to this active site. From experimental investigations it is known that 2-cis isomers are not substrates of the oligoprenyltransferase.  The resulting docking arrangement showed that the calculated affinity of the cis isomer to the enzyme (pKd(calc)=8.84) is reduced in comparison to the all trans isomer (pKd(calc)=10.11). However, more relevant is that the prenyl chain cannot be docked close to the 4-hydroxybenzoic acid due to significant steric interactions or even overlap with protein side chains. This result—also true for the other active site discussed—nicely explains why the (2-Z)-isomers of oligoprenyl diphosphates are not substrates.
A suggested mechanism for the prenylation of 4-hydroxybenzoic acid with oligoprenyl diphosphate catalyzed by 4-hydroxybenzoate oligoprenyltransferase (ubiA)
Based on our experimental substrate models and on the structure of active site 1 of the developed protein model, detailed investigations of the reaction mechanism were performed using semiempirical quantum mechanical PM3 calculations to analyze the catalytic mechanism.  For these calculations, all amino acid residues were simplified by using acetic acid or acetate to mimic aspartic acid or aspartate, respectively, lysine was represented by methylammonium, and arginine by methylguanidinium. Calculation distance constraints were set fixed for all carbon atoms of the methyl groups of the amino acid residue mimics to simulate the relative rigidity of the protein backbone and to ascertain the arrangement of the residues to each other. In analogy to common Mg2+ aspartate binding motifs, the magnesium ion forms a tetrahedral metal complex with Asp75 and the diphosphate of the dimethylallyl diphosphate (as a model for all prenyl diphosphates), and the carboxylic acid group of 4-hydroxybenzoate forms a salt bridge to the side chain of Arg137. Additionally, the orientation of the phenolic hydroxy group of the benzoic acid derivative is fixed by a hydrogen bond to Asp71.
After passing through the transition state, the reaction forms a σ complex intermediate (−800.6 kcal mol−1, cf. Fig. 6c). The C-1’–OPP distance for this optimized structure is 2.8 Å, the one from C-1’ to C-3 is 1.53 Å. A very small barrier of 4.7 kcal mol-1 has to be overcome to reach the final structures (−832.68 kcal mol−1, cf. Fig. 6d). Finally the C-1’ and diphosphate are separated by a distance of over 3.50 Å and the bond between C-1’ and C-3 is established with a distance of 1.49 Å. The transfer of the C-3 proton to the oxygen of the α-phosphate, which became accessible during the cleavage of the diphosphate, supposedly takes place simultaneously to the formation of the new C-1’–C-3 bond. This proton translocation requires no additional energy and no new energy barrier has to be passed.
Analysis of the substrate specificity by means of quantum mechanical investigations
Calculated ΔRH and ΔRH# values for benzoic acid substrates in kcal mol−1
It could be shown that some of these are viable substrates whereas others are not. Docking of some of the compounds to the active site 1 of the enzyme showed that in principal all could bind in an appropriate manner to allow catalysis. The only ligand that might bind in a slightly different orientation is 4-hydroxyphthalic acid. The difference is caused by the two neighboring carboxylic acids. This leads to an uncertain interaction with arginine137 and through this to the prenyl unit. Apart from this exception, it is obvious that electronic properties might play a dominant role for the substrate specificity. To explain the specificity, we performed semiempirical quantum mechanical calculations (PM3). First of all, the heats of formations of the phenols in comparison to the phenolates (first step in Scheme 2) were calculated. The results are listed in Table 4. From the thermodynamic point of view, it became evident that within the enzyme a phenolate anion and a neutral carboxylic acid unit are favored to the intuitively expected inverse deprotonation to a carboxylate and a phenol. The lowest energy gain is calculated for 4-amino-3-chloro-benzoic acid (formation of an imide anion).
This result, however, is of immediate importance for the catalysis, since a deprotonation of the phenolic group enhances the negative charge at the meta position of the 4-hydroxybenzoic acid by mesomeric stabilization and therefore will considerably support the reaction of the positively charged prenyl C-1’ with this position.
Finally, the energies of all intermediates of the pHB derivatives were calculated for the reaction as shown in Scheme 2. From the calculated energy difference between the σ complex and the educts it becomes obvious that the viable substrates are 4 to 9 kcal mol−1 favored in comparison to the derivatives which are not accepted as substrates. Since, the formation of prenylation products is thermodynamically favorable in all cases (last column in Table 4), the reactivity difference between substrates and non-substrates must have kinetic reasons caused by different transition state energies, or by steric interactions to fine too notice in our preliminary model. The special case of 4-hydroxyphthalic acid has already been discussed above.
A first 3D model of an oligoprenyltransferase has been developed by homology modeling based on an X-ray structure of the photosynthetic reaction center of Rhodopseudomonas viridis (1prc). Because of the very low amino acid identity, it is evident that this model is only a first crude approximation, hopefully with relevance for the real native structure, and thus has to be considered with care. Nevertheless, there is currently no better model available for aromatic prenyltransferases. The one presented here for the first time can help to understand principal mechanisms of substrate recognition and catalysis exhibited by this class of enzymes.
By means of multiple alignments, it could be shown that two putative active sites must be taken into consideration. Which of these, or if indeed both, are catalytically active must be validated by means of site-directed mutagenesis. However, in a review of Liang et al.  the effect of mutagenesis of the evolutionarily conserved aspartate residues in farnesyl diphosphate synthase is described. In a site-directed mutagensis experiment with yeast farnesyl diphosphate synthase, Song and Poulter , after a substitution of Asp by Ala, found kcat values with rates 4–5 orders of magnitude slower than the wild type. In the second xDDxxD motif of yeast, the first and second Asp to Ala mutations resulted in kcat values 4–5 orders of magnitude smaller. The third Asp of this motive seems to be less important to catalysis as its mutation to Ala only resulted in 6 to 16-fold lower kcat values.  Based on these results, Liang et al. conclude that all Asp residues in the two xDDxxD motifs except the last one in the second motif are important for catalysis. Our investigations are indirectly justified by this and it can be assumed that 4-hydroxybenzoate oligoprenyltransferase of E. coli is characterized by a similar mode of catalysis. However, since the ubiA enzyme only needs one diphosphate binding site, only one is likely active. The rudimentary second active site is a strong hint for the evolutionary origin of this aromatic prenyltransferase. It may have common roots with chain-elongation prenyl diphosphate synthases, which always need at least one more prenyl diphosphate binding site.
Furthermore, despite the uncertainties of the 3D model structure, some aspects of the substrate specificity could be explained based on the model. Based on the semiempirical quantum mechanical calculations it could be shown that some experimentally tested hydroxybenzoic acid derivatives are unsuitable as substrates due to high activation barriers during the catalysis. It could be shown that the whole process is thermodynamically favored and most likely proceeds via an SN1-type mechanism.
These results help to understand basic principals of the catalytic process exhibited by the ubiA enzyme and may serve as basis for the development of new ligands or inhibitors for this enzyme with its crucial importance for aerobic processes.