Inverse heavy enzyme isotope effects in methylthioadenosine nucleosidases

Heavy enzyme isotope effects occur in proteins substituted with 2H-, 13C-, and 15N-enriched amino acids. Mass alterations perturb femtosecond protein motions and have been used to study the linkage between fast motions and transition-state barrier crossing. Heavy enzymes typically show slower rates for their chemical steps. Heavy bacterial methylthioadenosine nucleosidases (MTANs from Helicobactor pylori and Escherichia coli) gave normal isotope effects in steady-state kinetics, with slower rates for the heavy enzymes. However, both enzymes revealed rare inverse isotope effects on their chemical steps, with faster chemical steps in the heavy enzymes. Computational transition-path sampling studies of H. pylori and E. coli MTANs indicated closer enzyme–reactant interactions in the heavy MTANs at times near the transition state, resulting in an improved reaction coordinate geometry. Specific catalytic interactions more favorable for heavy MTANs include improved contacts to the catalytic water nucleophile and to the adenine leaving group. Heavy bacterial MTANs depart from other heavy enzymes as slowed vibrational modes from the heavy isotope substitution caused improved barrier-crossing efficiency. Improved sampling frequency and reactant coordinate distances are highlighted as key factors in MTAN transition-state stabilization.

Acidic open-cage solution containing basic cage-confined nanospaces for multipurpose catalysis

The nanoscale chemical spaces inherent in various organic and metal-organic cages or porous solids and liquids have been continuously explored for their nanoconfinement effect on selective adsorption and reaction of small gas or organic molecules. Herein, we aim to rationalize the unconventional chemical reactivities motivated by the cage-confined nanospaces in aqueous solutions, where the robust yet permeable nanospaces defined by the open-cages facilitate dynamic guest exchange and unusual chemical reactions. The high positive charges on [(Pd/Pt)6(RuL3)8]28+ nanocages drive imidazole-proton equilibrium to display a significantly perturbed pKa shift, creating cage-defined nanospaces in solution with distinct intrinsic basicity and extrinsic acidity. The supramolecular cage effect plays pivotal roles in elaborating robust solution nanospaces, controlling ingress-and-egress molecular processes through open-cage portals, and endowing nanocages with transition-state stabilization, amphoteric reactivities and the phase transfer of insoluble molecules, thus promoting chemical transformations in unconventional ways. Consequently, a wide application of cage-confined catalysis with anomalous reactivities may be expected based on this kind of open-cage solution medium, which combines cage nanocavity, solution heterogeneity and liquid-phase fluidity to benefit various potential mass transfer and molecular process options.

Crystal structures of non-oxidative decarboxylases reveal a new mechanism of action with a catalytic dyad and structural twists

Hydroxybenzoic acids, like gallic acid and protocatechuic acid, are highly abundant natural compounds. In biotechnology, they serve as critical precursors for various molecules in heterologous production pathways, but a major bottleneck is these acids’ non-oxidative decarboxylation to hydroxybenzenes. Optimizing this step by pathway and enzyme engineering is tedious, partly because of the complicating cofactor dependencies of the commonly used prFMN-dependent decarboxylases. Here, we report the crystal structures (1.5–1.9 Å) of two homologous fungal decarboxylases, AGDC1 from Arxula adenivorans, and PPP2 from Madurella mycetomatis. Remarkably, both decarboxylases are cofactor independent and are superior to prFMN-dependent decarboxylases when heterologously expressed in Saccharomyces cerevisiae. The organization of their active site, together with mutational studies, suggests a novel decarboxylation mechanism that combines acid–base catalysis and transition state stabilization. Both enzymes are trimers, with a central potassium binding site. In each monomer, potassium introduces a local twist in a β-sheet close to the active site, which primes the critical H86-D40 dyad for catalysis. A conserved pair of tryptophans, W35 and W61, acts like a clamp that destabilizes the substrate by twisting its carboxyl group relative to the phenol moiety. These findings reveal AGDC1 and PPP2 as founding members of a so far overlooked group of cofactor independent decarboxylases and suggest strategies to engineer their unique chemistry for a wide variety of biotechnological applications.

Leptospira interrogans triosephosphate isomerase: exploring the structural determinants of stability, high reaction rate and specificity.

Leptospires are zoonotic pathogens that cause significant socio-economic burden in developing countries, world-wide. The pathogenic species Leptospira interrogans (Li) is an important and interesting target for investigating the enzymes essential to its metabolic needs and adaptations. We cloned and expressed triosephosphate isomerase (LiTIM), a central metabolic flux regulator of Li, in AA200, E. coli TIM null strain. LiTIM was obtained as an active dimer (D-GAP to DHAP, kcat = 1740 /s and Km (D-GAP) = 0.21 mM, at 25 C) with mid-transition concentrations, Cm, 0.8 mM and 2.6 mM, respectively, for guanidine hydrochloride and urea induced equilibrium unfolding. We report the high resolution X-ray structures of LiTIM in apo and substrate (DHAP) bound forms. Our analysis highlights key features of TIM that regulate the mode of substrate binding and transition state stabilization and thus play a decisive role in attainment of high proficiency for the isomerisation reaction while avoiding the elimination reaction. Unexpected differences in the effect of temperature on stability and activity were observed for the three mesophilic pathogenic TIMs viz. from Li, Plasmodium falciparum (Pf) and Trypanosoma brucei (Tb). LiTIM and TbTIM (Tm = 46.5 C) were more susceptible to unfolding and precipitation compared to PfTIM (Tm = 67 C). In contrast, the initial (or zero point) activity of PfTIM rises till 50 C and saturates unlike LiTIM and TbTIM which show a rise till 55 C and 60 C, respectively. These observations could be rationalized by sequence comparison and examination of the structures of the three TIMs.

How directed evolution reshapes the energy landscape in an enzyme to boost catalysis

The advent of biocatalysts designed computationally and optimized by laboratory evolution provides an opportunity to explore molecular strategies for augmenting catalytic function. Applying a suite of NMR, crystallographic, and stopped-flow techniques to an enzyme designed for an elementary proton transfer reaction, we show how directed evolution gradually altered the conformational ensemble of the protein scaffold to populate a narrow, highly active conformational ensemble and achieve a nearly billionfold rate acceleration. Mutations acquired during optimization enabled global conformational changes, including high-energy backbone rearrangements, that cooperatively organized the catalytic base and oxyanion stabilizer, thus perfecting transition-state stabilization. Explicit sampling of conformational sub-states during design, and specifically stabilizing productive over all unproductive conformations, could speed up the development of protein catalysts for many chemical transformations.

Understanding Enzymic Reactivity – New Directions and Approaches

New approaches towards understanding the reactivity of enzymes–central to chemical biology and a key to comprehending life itself–are discussed herein. The approach overall is based on the idea that structural and reactivity features uniquely characteristic of enzymes–in being absent in normal catalysts–are likely to hold the key to the catalytic powers of enzymes. The quintessentially physical-organic problem is addressed from several angles, both kinetic and phenomenological. (Generally, the Pauling theory of transition state stabilization is adopted as the rigorous basis for understanding enzyme action). The kinetic approach focuses on the inadequacies of the Michaelis-Menten equation, and proposes an alternative model based on additional substrate binding at high concentrations, which satisfactorily explains experimental observations. The phenomenological approaches focus on the inadequacies of the intramolecularity criterion, thus leading to alternative strategies adopted by nature in the design of these mild yet powerful catalysts, characterized by exquisite selectivity. Preferential transition state binding at the active site, via both hydrophobic and van der Waals forces, appears to be the major thermodynamic driver of enzymic reactivity. In operational terms, however, multifunctional catalysis–practically unique to the highly ordered enzyme interior–is likely the key to enzymic reactivity. A new concept, ‘strain delocalization’, possibly plays an important role in orchestrating these various effects, and indeed justifies the need for a large proteinic molecule for achieving the enormous rate enhancements generally observed with enzymes. Thus, this renewed approach to understanding enzymic reactivity departs significantly from currently held views: radically, in abandoning the Michaelis-Menten and intramolecularity models; but also commandeering existing ideas and concepts, although with a shift in emphasis towards transition state effects (including the entirely novel idea of ‘strain delocalization’).The coverage is not exhaustive, but aims to introduce new ideas along with fresh insights into previous works.