The (βα)8-barrel fold is the most frequently observed topology among enzymes. Due to the spatial separation of their activity- and stability-mediating sites, (βα)8-barrel enzymes are extremely versatile and catalyze a wide array of cellular reactions. Thus, this particular fold provides an ideal tool for modifying catalytic activity and studying enzyme evolution.
Several (βα)8-barrel enzymes are involved in the metabolism of amino acids. Along these lines, N’-[(5‘-phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide isomerase (HisA) and the cyclase subunit of imidazole glycerol phosphate synthase (HisF) catalyze two consecutive steps in the biosynthesis of histidine, namely a sugar isomerization and a cycloligase/lyase reaction. By analogy with HisA, the enzyme phosphoribosyl anthranilate isomerase (TrpF) performs a chemically equivalent isomerization reaction within the biosynthesis of tryptophan. As HisA, HisF, and TrpF accommodate their phosphorylated substrates via a common phosphate binding site, an evolutionary linkage seems to exist between these three (βα)8-barrel proteins. Consequently, HisA and HisF could be engineered to bind and process the TrpF substrate phosphoribosyl anthranilate (PRA). In both cases, a single aspartate-to-valine substitution was sufficient to establish PRA isomerase activity and the combination with a second aspartate-to-valine exchange significantly improved the turnover number. Since HisA and TrpF operate through an identical acid-base mechanism, the same enzymatic mechanism could be expected for the PRA isomerase activities generated on the HisA and HisF scaffolds. However, while mutational analyses and docking studies revealed a respective general base, no appropriate general acid could be identified. Therefore, the mechanistic foundation of the artificially designed PRA isomerase activity was addressed in the first part of this work.
Initially recorded pH dependences substantiated the mechanistic differences of the naturally occurring and the artificially designed PRA isomerase activity. While TrpF wild-type exhibited the expected bell-shaped pH profile, merely an acidic limb was observed for the investigated HisA variants, suggesting that the hitherto unidentified general acid is rate-limiting for the engineered reaction. A hint on the nature of the catalytic acid was subsequently obtained from the crystal structure of the HisF double mutant with a bound product analogue. The structure suggested that within the enzyme–substrate complex the anthranilic acid moiety of PRA might donate a proton to the furanose ring oxygen of its sugar moiety. This productive geometry of PRA was compared with a non-productive binding mode in molecular dynamics simulations of the HisA variants. In contrast to HisA wild-type, all variants clearly favor an orientation of PRA required for catalysis. Furthermore, the introduced valine residues clearly upshift the pKa value of anthranilic acid to a catalytically useful range. Mixed quantum and molecular mechanics calculations of the HisA double mutant with bound PRA finally demonstrated that an internal proton transfer is also feasible from an energetic point of view and presumably proceeds via a bridging water molecule. In sum, the artificial PRA isomerase activity established on the HisA and HisF scaffolds appears to be partly based on substrate-assisted catalysis and thus mechanistically deviates from the PRA isomerase activity of TrpF.
The second part of this dissertation dealt with the evolution of cellular complexity. Modern organisms are highly developed molecular machineries, which rely on elaborate enzyme systems. There is considerable interest to figure out which degree of enzymatic sophistication had already been reached in the very early phases of biological evolution. Along these lines, substantial progress has been made in the field of ancestral sequence reconstruction, which links computational and evolutionary biology and enables the characterization of extinct proteins. In extreme cases, enzymes from the last universal ancestor of cellular organisms (LUCA) can be studied. The LUCA preceded the diversification into the three domains of life and existed at least 3.5 billion years ago.
Exceptional catalytic features like substrate channeling and allosteric communication are observed in the imidazole glycerol phosphate synthase bi-enzyme complex of the cyclase HisF and the glutaminase HisH. In an attempt to analyze its primal characteristics, we reconstructed a HisF enzyme from the LUCA era (LUCA-HisF). LUCA-HisF could be expressed solubly in Escherichia coli and purified with a high yield. The protein furthermore shows a high thermostability and a folding mechanism comparable to extant (βα)8-barrel enzymes. Accordingly, its subsequently solved crystal structure equals contemporary HisF proteins. Beyond its structural integrity, LUCA-HisF proved to be a highly active and specific enzyme. As its catalytic sophis-tication could only be completely assessed in combination with an interacting gluta-minase, we additionally reconstructed a respective LUCA-HisH sequence. Although both proteins form a stoichiometric complex with high affinity, no catalytic activity could be determined for LUCA-HisH, probably due to uncertainties in the reconstruct-tion process. We instead turned to a complex between LUCA-HisF and the extant HisH enzyme from Zymomonas mobilis. Remarkably, LUCA-HisF could both stimulate the catalytic efficiency of the interacting glutaminase and transport the produced ammonia to its active site via a molecular channel. The evolution of these elaborate features therefore must already have been completed in the LUCA era.
The artificial control of enzymatic activity has been a long-standing goal in the field of protein design. Here, light provides an ideal trigger signal, since it enables a non-invasive and spatiotemporal regulation of biological activity. However, despite various attempts, only few enzymes have been successfully regulated by light so far. Therefore, the design of a light-controllable inhibitor of the (βα)8-barrel enzyme PriA from Mycobacterium tuberculosis (mtPriA) was the aim of the third section of this thesis. Interestingly, the bisubstrate-specific isomerase mtPriA is able to catalyze both the HisA and TrpF reaction and displays a potential target for anti-tuberculosis drugs, since humans can synthesize neither histidine nor tryptophan.
For the construction of the potential inhibitors, two particular features of mtPriA could be harnessed, both of which originate from the molecular evolution from a (βα)4-half-barrel precursor: the protein exhibits a striking twofold rotational symmetry as well as two opposite phosphate binding sites. Consequently, we chose the twofold symmetric, photoswitchable 1,2-dithienylethene (DTE) as a structural core and equipped it with terminal phosphate or phosphonate anchors. The synthesized DTE compounds could reversibly be toggled between a ring-open and ring-closed form by irradiation with UV and visible light, respectively. Both isomers were thermally stable, nearly quantitatively formed and robust over various switching cycles. When tested in steady-state enzyme kinetics, the open isomers of all DTE-phosphates and DTE-phosphonates competitively inhibited the mtPriA activity with the inhibition constants lying in the low micromolar range. Notably, the inhibition activity was lowered up to a factor of eight upon ring-closure, where the enzymatic performance could be directly controlled during catalysis. The different binding affinities obtained upon irradiation seem to be based on a change in the conformational flexibility. Along these lines, molecular dynamics simulations of mtPriA with an inhibitor bound in both isomeric forms demonstrated that the ring-open isomers can readily adapt to the active site of mtPriA. In contrast, due to its restricted mobility, the interaction of the ring-closed form with the enzyme is energetically less favorable. Thus, the dual anchoring of photoswitchable inhibitors constitutes a viable design concept for the reversible regulation of enzymatic activity. The approach may additionally be transferred to other (βα)8-barrel proteins, as phosphate is a frequently encountered element of metabolic substrates
