Fluorescence Spectroscopy Investigation of the Enzymatic Mechanism of Phytochelatin Synthase, an Enzyme Able to Cleave and Form Peptide Bond
Final Report Abstract
Phytochelatin synthase (PCS) catalyzes the non-ribosomal formation of the metal binding peptide phytochelatin (PCn, (γGlu-Cys)nGly) from glutathione (GSH, γGlu-Cys-Gly). The catalytic mechanism of PCS leading to the formation of phytochelatins consists in: (i) peptidase activity - the cleavage of the glycine from GSH, (ii) transpeptidase activity - the formation of a peptide bond between the resulting γ-glutamyl-cysteine dipeptide and another GSH molecule. To analyze the molecular mechanism of PCS, we designed FRET-based experiments demanding appropriate fluorescently-labeled and chemically-modified GSH analogues. During the funded period, we developed a potent scheme of synthesis, which allows producing the desired GSH analogues from precursory building blocks, following the strategy of different protection groups. The relevance of this achievement goes beyond the specific goals of this project, because glutathione (GSH) is an essential molecule, which is present in every cells, from prokaryotes to eukaryotes; besides being an important antioxidant, GSH is also substrate of several vital enzymes. Therefore, the straightforward approach of engineering GSH that we have developed is of paramount importance to dissect the molecular mechanism of GSH-dependent enzymes and to further understand the functions of this ubiquitous molecule. To allow investigation with optical spectroscopy methods, GSH analogues labeled with appropriate chromophores were synthesized as well. Unexpectedly, the commercially available and widely used fluorescence quencher dabcyl that we selected to be the acceptor in our FRET-based experiments perturbed the enzymatic activity of PCS preventing its investigation. Assessed that this inconvenient is mainly due to the hydrophobicity of the molecule, we designed, synthetized and characterized a novel fluorescence quencher – hydrodabcyl – corresponding to a hydrophilic analogue of dabcyl. This new molecule represents an improved alternative to the very popular dabcyl in the design of fluorogenic probes and has been patented. In many enzymatic reactions in which GSH acts as a substrate, its amide bonds are often cleaved or formed, as in the reaction catalyzed by PCS. In order to gain further insight into these processes, we also explored several options of non-cleavable amide bond isosters. During our quest for tools to investigate different specific steps of the enzymatic mechanism of PCS, we designed and synthetized a plethora of chemically modified GSH analogues and we could identified also a potential inhibitor. The initially unplanned access to crystallography, prompted us to solve high-resolution crystal structures of the native and specifically mutated forms of PCS from the cyanobacterium Nostoc sp. Strain PCC 712 (NsPCS). The molecular structures that are now in our hand constitute an ideal basis for theoretical calculations, which are essential for interpreting experimental results on structural basis as well as predicting properties that can be then tested experimentally. The results and the tools that we have developed along this funding period are extremely helpful to proceed with the investigation of PCS and are also expected to boost the investigation of many other GSH-dependent enzymes.
Publications
- Methods based on continuum electrostatics and their application to flavoproteins. In Handbook of Flavoproteins, Vol. 2. (S. Miller, B. Palfey and R. Hille, Eds), De Gruyter, Berlin, 2013, 335-360
Ullmann G. M., Dumit V. I. & Bombarda E.
- pKa values and redox potential of proteins. What do they mean? Biol. Chem. 2013, 394, 611–619
Ullmann G. M. & Bombarda E.
(See online at https://doi.org/10.1515/hsz-2012-0329) - Continuum electrostatic analysis of protein. In Protein Modelling (G. Náray-Szabó, Ed.), Springer International Publishing Switzerland, 2014, 135- 163
Ullmann G. M. & Bombarda E.
- Theoretical analysis of electrostatics in proteins. From simple proteins to complex machineries. In Advances in Photosynthesis and Respiration, Vol. 41. Cytochrome Complexes: Evolution, Structures, Energy transduction and Signaling. (W. A. Cramer and T. Kallas Eds.), Springer Verlag, 2016, 99-127
Ullmann G. M., Müller L. & Bombarda E.
