Chapter 3. Results and Discussion
3.6 Catechol derivatives oxidative activity of copper-substituted PepD (CuCu-PepD)
In our previous study, the hydrolysis activity of PepD was found to be inhibited by dopamine or L-dopa. Interestingly, the peptide-bond hydrolysis activity of PepD can be converted to catechol oxidative activity by substitution of zinc ions with copper ions in active site (CuCu-PepD). However, even though CuCu-PepD can oxidize catechol derivatives, which are catecholamine hormones produced from tyrosine metabolism (dopamine, L-dopa, epinephrine and norepinephrine) (Fig. 3.13), it is unable to oxidize catechol or 3,5-di-tert-butylcatechol (DTC), which has distinguished by their non-polar side chains (Fig. 3.14). This result indicated that CuCu-PepD specifically reacts with only substrates having a polar tail. No other enzyme to date, to our knowledge, has been reported to have such a property of specialized substrate selectivity.
Furthermore, enzyme kinetics analysis revealed similar catalytic efficiency (kcat/Km) of CuCu-PepD for several catecholamine hormones. CuCu-PepD oxidation of epinephrine was more efficient than that of other related substrates (Table 3.4). This observation likely reflects the good binding that exists between enzyme and epinephrine (Km = 0.073 mM). Comparison of the enzyme kinetics between CuCu-PepD and copper-substituted SgAP (CuCu-SgAP), as reported by Ming et al.72, indicated that CuCu-PepD exhibited better substrate binding affinity for dopamine (Km = 0.6 mM) and DTC (Km = 0.44 mM) than did CuCu-SgAP. However, the low kcat of CuCu-PepD for several catechol derivatives leads to decreased oxidative efficiency (Table 3.4).
67
Figure 3.13. Catecholamine hormones produced from tyrosine metabolism.
Figure 3.14. Structures of catechol and its derivatives. The polar tail of catechol derivatives are indicated by red color.
68
Table 3.4: Comparison of enzyme kinetics of PepD, CuCu-PepD, and CuCu-SgAP*
for different substrates. *Referred from Ming et al.72 (gray background).
3.7 Protein-ligand docking between CuCu-PepD and catecholamine hormones (L-dopa, dopamine, epinephrine, and norepinephrine)
In the previous report by Ming et al., the active site of CuCu-SgAP provides the hydrophobic pocket for specific recognition in binding of DTC via its di-tert-butyl groups. However, CuCu-PepD was unable to oxidize catechol or DTC, but it could oxidize the catechol derivatives with polar tail. In order to understand the binding network for each catecholamine hormones with CuCu-PepD, a protein-ligand docking strategy was applied to investigate the molecular interaction that occurred between enzyme and substrate. Based on the results obtained, dopamine, L-dopa, epinephrine, and norepinephrine could be bound by CuCu-PepD in the enzyme’s metal ions binding center. The docking model of the CuCu-PepD-dopamine complex indicated that ortho-hydroxyl group of dopamine could be bound by Asp119, Glu149, Asp173 and His461, and the amine group could be bound by the Glu175 side chain via hydrogen bonding (Fig.
3.15A). In addition, the binding network of L-dopa with CuCu-PepD was found to be similar to that for dopamine (Fig. 3.15B). Since the amine group of L-dopa is distant
69
from Glu175, the combination between the hydroxyl group of Tyr181 and the carboxylate group of L-dopa is more likely (Fig. 3.15B). When the binding networks between epinephrine and norepinephrine were compared, they were found to be similar in that hydroxyl groups of both substrates were bound by Glu175 at a distance of 2.7 Å (Fig.
3.15 C and D). However, the end-methyl group of epinephrine was able to further interaction via the hydrophobic pocket produced by Ile432 and Ala434. Therefore, the protein-ligand docking results indicated that the better catalytic efficiency of CuCu-PepD for epinephrine was a result of epinephrine fitting better into the PepD active site.
Figure 3.15. Protein-ligand docking of CuCu-PepD with (A) dopamine, (B) L-dopa, (C) epinephrine, and (D) norepinephrine. A local view of the di-metal ions center of PepD is presented for each. Residues are shown as sticks and labeled accordingly. The copper ions of CuCu-PepD are depicted by two light blue spheres.
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CHAPTER 4
Conclusions and Future Perspectives
In conclusion, despite the lack of detectable sequence homology, the PepD enzyme has clear structural homology to other di-zinc-dependent M20 and M28 family enzymes. The crystal structure of V. alginolyticus PepD reveals it to be a dimer with two domains in each subunit. The catalytic domain of PepD contains two zinc ions and is structurally homologous to other proteolytic enzymes with dinuclear zinc catalytic sites.
The lid domain, on the other hand, is structurally homologous to that of PepV, but having a distinct topology of β-sheet order. Interestingly, part of the lid domain of the PepD structure is also homologous to the lid domain of the dimeric proteins.
Nevertheless, PepV exists as a monomer, while the PepD and related di-zinc-dependent M20/M28 family of enzymes are determined to be dimers. Structural comparisons between PepD and related di-zinc-dependent metallopeptidases suggest that formation of the catalytically competent active site in the PepD family of enzymes may be associated with transition from an open to a closed enzyme conformation. In parallel, the site-directed mutation of the putative substrate C-terminal binding residues, N260A and R369A, resulted in complete loss or partial decrease of the enzymatic activity.
Furthermore, enzymatic assay of the truncated PepD catalytic domain, PepDCAT, further demonstrated the functional role of the lid domain in substrate binding and selectivity.
The structural data on PepD reported here may inspire strategies for the improvement of the PepD family of enzymes toward applications in biotechnology and allow the design of targeted disease peptidases or prodrugs with altered specificity.
71
On the other hand, it is important to consider the observation that substitution of zinc ions with copper ions in the PepD active site generated an enzyme, CuCu-PepD, preferred catechol derivatives containing polar tails. Based on the model of protein-ligand docking, the ortho-hydroxyl groups of each substrate are bound by Asp119, Glu149, Asp173 and His461 through hydrogen bonding as well as through polar tail interactions with Glu175 and Tyr181. The model of CuCu-PepD-epinephrine fitted better into the active site by additional hydrophobic interactions between the PepD Ile432 and Ala434 and the terminal methyl group of epinephrine. Enzyme kinetics studies indicated that oxidation of epinephrine produced notable catalytic efficiency, explaining its low Km value. In all, PepD is a unique enzyme, whose function can be effectively altered by simple metal ions substitution. This study not only unveiled a correlation of enzyme function between peptide hydrolysis and catechol oxidation, but also may provide a new direction by which divergent enzyme evolution occurs.
There are several possible directions that could be considered for future studies based upon the findings presented here. To better understand the PepD structure-function relationship and biological function, and to determine potential applications in enzymatic therapy, the following investigations are suggested:
I. Structure analysis of PepD-inhibitor complex by X-ray crystallography
In our previous report, the crucial residues in PepD for metal ions binding and substrate C-terminal and/or transition state binding were identified. We used PepD native structure and mutational analysis; however, the substrate binding network and substrates can induce conformational changes in the enzyme that were not addressed by our study. Therefore, co-crystallization of PepD with inhibitor (bestatin or dopamine) would provide further insights into the substrate binding sites and catalytic mechanism.
72
Furthermore, the enzyme conformational change from the open to closed form might also be determined by complex structure.
II. Proteomic study of V. alginolyticus pepD-deficient knockout strain
Proteomic study includes not only the identification and quantification of a particular protein panel under certain physiologic conditions, but also the determination of their localization, modifications, interactions, activities, and ultimately their function.
A previous report by Brombacher et al., in which pepD gene was overexpressed in E.
coli, indicated that PepD was vital to reducing biofilm formation23. In a similar manner, a V. alginolyticus pepD-deficient strain might be used to investigate morphology and biofilm formation by comparing with wild-type. Two-dimensional protein electrophoresis and mass spectrometry would help to provide more detailed proteomic information about the biological effect on protein signaling pathways by PepD.
III. Potential application in antibody directed enzyme prodrug therapy (ADEPT) In ADEPT, the range of potential enzymes is limited by several constraints. For example, the enzyme’s function is to efficiently convert an inactive prodrug into the active drug; thus, its optimal pH must be near the pH of the tumor extracellular fluid.
V. alginolyticus PepD considered a promising candidate for use in ADEPT because it works at pH 6.8-7.4, which is similar to the environment of human body. In addition, it can hydrolyze a dipeptide into two amino acids, which is affectively the same mechanism of prodrug hydrolyzation. The analysis of V. alginolyticus PepD performed to date (including functional residues characterization, protein structure, mutagenesis analysis and enzyme kinetics analysis) would aid in the design of a PepD mutant with improved digestion efficiency for a particular prodrug with a dipeptide
73
skeleton. Therefore, it is feasible that enzymatic engineering of PepD can generate desired mutant proteins to hydrolyze a prodrug, such as benzoic acid mustard prodrug (Fig. 1.9). This type of PepD study will require the development of an animal model to determine the efficacy and safety of a PepD-based ADEPT strategy, prior to its use in humans.
IV. Molecular evolution of metal-substituted dipeptidase for protein plasticity
A unique enzyme catalytic promiscuity has recently been observed for the dinuclear aminopeptidase from Streptomyces griseus. SgAP exhibits a high efficiency of catalytic promiscuity toward phosphonate and phosphodiester hydrolysis under different physiological conditions67, 68. Moreover, the peptide hydrolysis activity of SgAP70, 71 could be converted to catechol oxidative activity by manipulation of its metal derivatives72.
In PepD, we have observed catechol oxidative activity induced by copper ions substitution in the active site. Using the crystal structure of PepD and attaining a better understanding of the critical role of individual amino-acid residues involved in metal ions binding and substrate recognition, it may be possible to create an artificial enzyme with phosphoester hydrolysis activity. This might be achieved by a combination of site-directed mutagenesis and metal substitution of PepD protein. Development of an artificial PepD exhibiting phosphoesterase function might generate new potentials in chemical warfare, such as degradation of the nerve agent Soman toxin89-91.
74
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