Analisi cinetiche, spettrofotometriche e mutazionali di due enzimi PLP-dipendenti del fold type I. Meccanismi catalitici, processi di folding e dimerizzazione

The results described in this thesis concern the structure-functional relationships of two PLPdependent enzyme belonging to the fold-type I family: 1) Treponema denticola cystalysin and 2) human alanine:glyoxylate amonotransferase (AGT). 1) Cystalysin, a key virulence factor of the human oral pathogen Treponema denticola responsible for periodontis, is a homodimeric pyridoxal 5’-phosphate (PLP)-dependent enzyme, which catalyzes the α,β-elimination of L-cysteine producing pyruvate, ammonia, and H2S. Cystalysin toxicity has been related to the production of H2S, a compound that is toxic to most cells at high concentrations. Previews investigations carried out by my research group provided evidence for remarkable difference in the near-UV CD spectra of holo and apo-cystalysin, and in their ability to enhance ANS emission fluorescence and intensity. In order to gain further insights on this issue, in this study the role of PLP cofactor in the structural features, and in both folding and dimerization processes of the enzyme has been investigated. Several indicators have been used as probes of the different conformational status of T. denticola cystalysin in the holo and apo form. Compared to holoenzyme, the apoenzyme displays an altered reactivity of cysteine residues, a significantly different pI, and a differential susceptibility to proteinase K. These findings confirmed that apo and holocystalysin represent two distinct conformational states of the enzyme. In addiction, the urea-induced folding equilibrium of holo and apocystalysin has been studies by a variety of biophysical techniques and analytical procedures. Urea-induced unfolding profiles show that monomerization of apocystalysin occurs at low denaturant concentration without the significant loss of secondary and tertiary structures, whereas it takes place for holocystalysin at high denaturant concentration, once the structure is sufficiently destabilized. Moreover, refolding studies, together with analysis of the dissociation/association process of cystalysin, shed light on how the protein concentration and the presence or absence of PLP under refolding conditions could affect the dimerization of the enzyme and the production of insoluble aggregates. In order to clarify the role of the cofactor in the dimerization process, two interfacial residues (Leu57 and Leu62) and an active site residue (Tyr64*), hydrogen-bonded with the PLP phosphate group of the neighboring subunit, have been mutated on the base of preliminary bioinformatic analysis. The wild-type and the L57A, L62A, Y64*A, L57A/L62A, L57A/Y64*A, L57A/L62A/Y64*A mutants have been constructed, expressed, and purified. The impact of these mutations on the dimeric state of cystalysin in the apo- and holo-form has been analyzed by sizeexclusion chromatography. The results shed light on the role of Leu57, Leu62 and Tyr64 in the dimerization process of holo and apoenzyme, and demonstrate that combination of two or three mutations leads to species which remain in a folded monomeric state at a reasonably high concentration in both the apo- and holo-forms. In particular L57A/L62A/Y64*A remains prevalently monomer at a concentration up to 50 μM. Kinetic analyses show that, in monomeric triple mutant, the α,β-eliminase, alanine racemase, and D-alanine half-transaminase activities are almost abolished, while the L-alanine half-transaminase activity is slightly enhanced when compared with that of wild-type cystalysin. The structural basis of the stereospecific transaminase activity displayed by the engineered folded PLP-bound monomer has been analyzed and discussed. 2) Human hepatic peroxisomal AGT (alanine:glyoxylate aminotransferase) is a PLP (pyridoxal 5’- phosphate)-dependent enzyme whose deficiency causes primary hyperoxaluria type I, a rare autosomal recessive disorder. To acquire experimental evidence for the physiological function of AGT, the Keq,overall of the reaction, the steady-state kinetic parameters of the forward and reverse reactions, and the pre-steady-state kinetics of the half-reactions of the PLP form of AGT with Lalanine or glycine and the PMP (pyridoxamine 5’-phosphate) form with pyruvate or glyoxylate have been measured. The results indicate that the enzyme is highly specific for catalysing glyoxylate to glycine processing, thereby playing a key role in glyoxylate detoxification. Analysis of the reaction course also reveals that PMP remains bound to the enzyme during the catalytic cycle and that the AGT–PMP complex displays a reactivity towards oxo acids higher than that of apoAGT in the presence of PMP. These findings are tentatively related to possible subtle rearrangements at the active site also indicated by the putative binding mode of catalytic intermediates. Additionally, the catalytic and spectroscopic features of the naturally occurring G82E variant have been analysed. Although, like the wild-type, the G82E variant is able to bind 2 mol PLP/dimer, it exhibits a significant reduced affinity for PLP and even more for PMP compared with wild-type, and an altered conformational state of the bound PLP. The striking molecular defect of the mutant, consisting in the dramatic decrease of the overall catalytic activity (0.1% of that of normal AGT), appears to be related to the inability to undergo an efficient transaldimination of the PLP form of the enzyme with amino acids as well as an efficient conversion of AGT–PMP into AGT–PLP. Overall, careful biochemical analyses have allowed elucidation of the mechanism of action of AGT and the way in which the disease causing G82E mutation affects it