A Lumenal Loop Associated with Catalytic Asymmetry in Plant Vacuolar H+-Translocating Pyrophosphatase

Membrane-integral inorganic pyrophosphatases (mPPases) couple pyrophosphate hydrolysis with H+ and Na+ pumping in plants and microbes. mPPases are homodimeric transporters with two catalytic sites facing the cytoplasm and demonstrating highly different substrate-binding affinities and activities. The structural aspects of the functional asymmetry are still poorly understood because the structure of the physiologically relevant dimer form with only one active site occupied by the substrate is unknown. We addressed this issue by molecular dynamics (MD) simulations of the H+-transporting mPPase of Vigna radiata, starting from its crystal structure containing a close substrate analog (imidodiphosphate, IDP) in both active sites. The MD simulations revealed pre-existing subunit asymmetry, which increased upon IDP binding to one subunit and persisted in the fully occupied dimer. The most significant asymmetrical change caused by IDP binding is a ‘rigid body’-like displacement of the lumenal loop connecting α-helices 2 and 3 in the partner subunit and opening its exit channel for water. This highly conserved 14–19-residue loop is found only in plant vacuolar mPPases and may have a regulatory function, such as pH sensing in the vacuole. Our data define the structural link between the loop and active sites and are consistent with the published structural and functional data.

Mechanistic basis for the emergence of EPS1 as a catalyst in plant salicylic acid biosynthesis

Unique to plants in the Brassicaceae family, the production of the plant defense hormone salicylic acid (SA) from isochorismate is accelerated by an evolutionarily young isochorismoyl-glutamate pyruvoyl-glutamate lyase, EPS1, which belongs to the BAHD acyltransferase protein family. Here, we report the crystal structures of apo and substrate-analog-bound EPS1 from Arabidopsis thaliana. Assisted by microsecond molecular dynamics simulations, we uncover a unique pericyclic rearrangement lyase mechanism facilitated by the active site of EPS1. We reconstitute the isochorismate-derived pathway of SA biosynthesis in Saccharomyces cerevisiae, which serves as an in vivo platform that helps identify active-site residues critical for EPS1 activity. This study describes the birth of a new catalyst in plant phytohormone biosynthesis by reconfiguring the ancestral active site of a progenitor enzyme to catalyze alternative reaction.One sentence summaryBy reconfiguring the active site of a progenitor acyltransferase-fold, EPS1 acquired the unique, evolutionarily new lyase activity that accelerates phytohormone salicylic acid production in Brassicaceae plants.

Gcn5-Related N-Acetyltransferases (GNATs) With a Catalytic Serine Residue Can Play Ping-Pong Too

Enzymes in the Gcn5-related N-acetyltransferase (GNAT) superfamily are widespread and critically involved in multiple cellular processes ranging from antibiotic resistance to histone modification. While acetyl transfer is the most widely catalyzed reaction, recent studies have revealed that these enzymes are also capable of performing succinylation, condensation, decarboxylation, and methylcarbamoylation reactions. The canonical chemical mechanism attributed to GNATs is a general acid/base mechanism; however, mounting evidence has cast doubt on the applicability of this mechanism to all GNATs. This study shows that the Pseudomonas aeruginosa PA3944 enzyme uses a nucleophilic serine residue and a hybrid ping-pong mechanism for catalysis instead of a general acid/base mechanism. To simplify this enzyme’s kinetic characterization, we synthesized a polymyxin B substrate analog and performed molecular docking experiments. We performed site-directed mutagenesis of key active site residues (S148 and E102) and determined the structure of the E102A mutant. We found that the serine residue is essential for catalysis toward the synthetic substrate analog and polymyxin B, but the glutamate residue is more likely important for substrate recognition or stabilization. Our results challenge the current paradigm of GNAT mechanisms and show that this common enzyme scaffold utilizes different active site residues to accomplish a diversity of catalytic reactions.

Crystal structure of inhibitor-bound human MSPL that can activate high pathogenic avian influenza

Infection of certain influenza viruses is triggered when its HA is cleaved by host cell proteases such as proprotein convertases and type II transmembrane serine proteases (TTSP). HA with a monobasic motif is cleaved by trypsin-like proteases, including TMPRSS2 and HAT, whereas the multibasic motif found in high pathogenicity avian influenza HA is cleaved by furin, PC5/6, or MSPL. MSPL belongs to the TMPRSS family and preferentially cleaves [R/K]-K-K-R↓ sequences. Here, we solved the crystal structure of the extracellular region of human MSPL in complex with an irreversible substrate-analog inhibitor. The structure revealed three domains clustered around the C-terminal α-helix of the SPD. The inhibitor structure and its putative model show that the P1-Arg inserts into the S1 pocket, whereas the P2-Lys and P4-Arg interacts with the Asp/Glu-rich 99-loop that is unique to MSPL. Based on the structure of MSPL, we also constructed a homology model of TMPRSS2, which is essential for the activation of the SARS-CoV-2 spike protein and infection. The model may provide the structural insight for the drug development for COVID-19.

Structure and kinetics of indole-3-glycerol phosphate synthase from Pseudomonas aeruginosa: Decarboxylation is not essential for indole formation

In tryptophan biosynthesis, the reaction catalyzed by the enzyme indole-3-glycerol phosphate synthase (IGPS) starts with a condensation step in which the substrate's carboxylated phenyl group makes a nucleophilic attack to form the pyrrole ring of the indole, followed by a decarboxylation that restores the aromaticity of the phenyl. IGPS from Pseudomonas aeruginosa has the highest turnover number of all characterized IGPS enzymes, providing an excellent model system to test the necessity of the decarboxylation step. Since the 1960s, this step has been considered to be mechanistically essential based on studies of the IGPS–phosphoribosylanthranilate isomerase fusion protein from Escherichia coli. Here, we present the crystal structure of P. aeruginosa IGPS in complex with reduced CdRP, a nonreactive substrate analog, and using a sensitive discontinuous assay, we demonstrate weak promiscuous activity on the decarboxylated substrate 1-(phenylamino)-1-deoxyribulose-5-phosphate, with an ∼1000× lower rate of IGP formation than from the native substrate. We also show that E. coli IGPS, at an even lower rate, can produce IGP from decarboxylated substrate. Our structure of P. aeruginosa IGPS has eight molecules in the asymmetric unit, of which seven contain ligand and one displays a previously unobserved conformation closer to the reactive state. One of the few nonconserved active-site residues, Phe201 in P. aeruginosa IGPS, is by mutagenesis demonstrated to be important for the higher turnover of this enzyme on both substrates. Our results demonstrate that despite IGPS's classification as a carboxy-lyase (i.e. decarboxylase), decarboxylation is not a completely essential step in its catalysis.

Energetics and kinetics of substrate analog-coupled staphylococcal nuclease folding revealed by a statistical mechanical approach

Protein conformational changes associated with ligand binding, especially those involving intrinsically disordered proteins, are mediated by tightly coupled intra- and intermolecular events. Such reactions are often discussed in terms of two limiting kinetic mechanisms, conformational selection (CS), where folding precedes binding, and induced fit (IF), where binding precedes folding. It has been shown that coupled folding/binding reactions can proceed along both CS and IF pathways with the flux ratio depending on conditions such as ligand concentration. However, the structural and energetic basis of such complex reactions remains poorly understood. Therefore, we used experimental, theoretical, and computational approaches to explore structural and energetic aspects of the coupled-folding/binding reaction of staphylococcal nuclease in the presence of the substrate analog adenosine-3′,5′-diphosphate. Optically monitored equilibrium and kinetic data, combined with a statistical mechanical model, gave deeper insight into the relative importance of specific and Coulombic protein–ligand interactions in governing the reaction mechanism. We also investigated structural aspects of the reaction at the residue level using NMR and all-atom replica-permutation molecular dynamics simulations. Both approaches yielded clear evidence for accumulation of a transient protein–ligand encounter complex early in the reaction under IF-dominant conditions. Quantitative analysis of the equilibrium/kinetic folding revealed that the ligand-dependent CS-to-IF shift resulted from stabilization of the compact transition state primarily by weakly ligand-dependent Coulombic interactions with smaller contributions from specific binding energies. At a more macroscopic level, the CS-to-IF shift was represented as a displacement of the reaction “route” on the free energy surface, which was consistent with a flux analysis.