Biochemistry

InhA, the Mycobacterium tuberculosis enoyl-ACP reductase, is a target for the tuberculosis (TB) drug isoniazid (INH). InhA inhibitors that do not require KatG activation avoid the most common mechanism of INH resistance, and there are continuing efforts to fully elucidate the enzyme mechanism to drive inhibitor discovery. InhA is a member of the short-chain dehydrogenase/reductase superfamily characterized by a conserved active site Tyr, Y158 in InhA. To explore the role of Y158 in the InhA mechanism, this residue has been replaced by fluoroTyr residues that increase the acidity of Y158 up to ∼3200-fold...

RNA enzymes (ribozymes) often rely on specific base-pairing interactions to engage RNA substrates, which limits the substrate sequence generality of these enzymes. An RNA polymerase ribozyme that was previously optimized by directed evolution to operate in a more efficient and sequence-general manner can now recognize the RNA template, RNA primer, and incoming nucleoside 5'-triphosphate (NTP) entirely through tertiary interactions. As with proteinaceous polymerases, these tertiary interactions are largely agnostic to the sequence of the template, which is an essential property for the unconstrained transmission of genetic information...

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that catalyze oxidative cleavage of polysaccharides, such as cellulose and chitin. LPMO catalysis requires a reductant, such as ascorbic acid, and hydrogen peroxide, which can be generated in situ in the presence of molecular oxygen and various electron donors. While it is known that reduced LPMOs are prone to autocatalytic oxidative damage due to off-pathway reactions with the oxygen co-substrate, little is known about the structural consequences of such damage...

In eukaryotic cells, the subcellular targeting of RNA controls many fundamental aspects of cellular physiology. Despite broad distributions throughout the cytoplasm, RNA molecules are conventionally believed to be excluded from the secretory pathway compartments including the endoplasmic reticulum (ER). Recent discovery of RNA N -glycan modification (glycoRNAs) has challenged this view, but direct evidence of RNA localization in the ER lumen has been lacking. In this study, we applied enzyme-mediated proximity labeling to profile the ER lumen-localized RNAs in human embryonic kidney 293T cells and rat cortical neurons...

From its structure and mechanism, sucrose phosphorylase is a specialized glycoside hydrolase that uses phosphate ions instead of water as the nucleophile of the reaction. Unlike the hydrolysis reaction, the phosphate reaction is readily reversible and, here, this has enabled the study of temperature effects on kinetic parameters to map the energetic profile of the complete catalytic process via a covalent glycosyl enzyme intermediate. Enzyme glycosylation from sucrose and α-glucose 1-phosphate (Glc1P) is rate-limiting in the forward ( k cat = 84 s-1 ) and reverse direction ( k cat = 22 s-1 ) of reaction at 30 °C...

The initial stage of fibril formation of C-terminal region PAP(248-286) of human seminal plasma protein prostatic acid phosphatase was considered. Amyloid fibrils from the peptide PAP(248-286) are termed as a semen-derived enhancer of viral infection (SEVI) found in abundant quantities in semen. The kinetics of the amyloid fibril formation process consists of two characteristic phases (lag phase/nucleation phase and growth phase/elongation phase). The lag phase can be caused by the presence of mature amyloid fibrils (seeds) in protein solution, so-called secondary nucleation...

The mechanism of protein aggregation can be broadly viewed as a shift from the native-state stabilizing intramolecular to the aggregated-phase sustaining intermolecular interactions. Understanding the role of electrostatic forces on the extent of modulation of this switch has recently evolved as a topic of monumental significance as protein aggregation has lately been connected to charge modifications of an aging proteome. To decipher the distinctive role of electrostatic forces on the extremely complicated phase separation landscape, we opted for a combined in vitro-in silico approach to ascertain the structure-dynamics-stability-aggregability relationship of the functional tandem RRM domains of the ALS-related protein TDP-43 (TDP-43tRRM ), under a bivariate solution condition in terms of pH and salt concentration...

Microbial rhodopsins are light-receptive proteins with various functions triggered by the photoisomerization of the retinal chromophore from the all- trans to 13- cis configuration. A retinal chromophore is covalently bound to a lysine residue in the middle of the seventh transmembrane helix via a protonated Schiff base. Bacteriorhodopsin (BR) variants lacking a covalent bond between the side chain of Lys-216 and the main chain formed purple pigments and exhibited a proton-pumping function. Therefore, the covalent bond linking the lysine residue and the protein backbone is not considered a prerequisite for microbial rhodopsin function...

Several proteins have been shown to undergo a shift in the mechanism of ligand binding-induced folding from conformational selection (CS; folding precedes binding) to induced fit (IF; binding precedes folding) with increasing ligand concentration. In previous studies of the coupled folding/binding reaction of staphylococcal nuclease (SNase) in the presence of a substrate analogue, adenosine-3',5'-diphosphate (prAp), we found that the two phosphate groups make important energetic contributions toward stabilizing its complex with the native protein as well as transient conformational states encountered at high ligand concentrations favoring IF...

Alpha hemolysin of Escherichia coli (HlyA) is a pore-forming protein, which is a prototype of the " R epeat in T o x ins" (RTX) family. It was demonstrated that HlyA-cholesterol interaction facilitates the insertion of the toxin into membranes. Putative cholesterol-binding sites, called cholesterol recognition/amino acid consensus (CRAC), and CARC (analogous to CRAC but with the opposite orientation) were identified in the HlyA sequence. In this context, two peptides were synthesized, one derived from a CARC site from the insertion domain of the toxin (residues 341-353) (PEP 1) and the other one from a CRAC site from the domain between the acylated lysines (residues 639-644) (PEP 2), to study their role in the interaction of HlyA with membranes...

The ERM (ezrin, radixin, and moesin) family of proteins and the related protein merlin participate in scaffolding and signaling events at the cell cortex. The proteins share an N-terminal FERM [band four-point-one (4.1) ERM] domain composed of three subdomains (F1, F2, and F3) with binding sites for short linear peptide motifs. By screening the FERM domains of the ERMs and merlin against a phage library that displays peptides representing the intrinsically disordered regions of the human proteome, we identified a large number of novel ligands...

SARS-CoV-2 continues to pose a threat to public health. Main protease (Mpro ) is one of the most lucrative drug targets for developing specific antivirals against SARS-CoV-2 infection. By targeting Mpro , peptidomimetic nirmatrelvir is able to inhibit viral replication of SARS-CoV-2 and reduce the risk for progression to severe COVID-19. However, multiple mutations in the gene encoding Mpro of emerging SARS-CoV-2 variants raise a concern of drug resistance. In the present study, we expressed 16 previously reported SARS-CoV-2 Mpro mutants (G15S, T25I, T45I, S46F, S46P, D48N, M49I, L50F, L89F, K90R, P132H, N142S, V186F, R188K, T190I, and A191V)...

Peroxiredoxins (Prxs) belong to a family of ubiquitously expressed peroxidases that detoxify reactive oxygen species. In addition to their enzymatic function, Prxs also function as molecular chaperones. This functional switch is related to their degree of oligomerization. We have previously revealed that Prx2 interacts with anionic phospholipids and that the anionic phospholipid-containing Prx2 oligomer forms a high molecular weight (HMW) complex in a nucleotide-dependent manner. However, the detailed mechanism of the oligomer and HMW complex formation remains unclear...

The molecular details of the interaction between human angiogenin (hAng) and proliferating cell nuclear antigen (PCNA) have been investigated by isothermal titration calorimetry (ITC), mutagenesis, and NMR spectroscopy. The two proteins were shown to interact directly through immunoprecipitation studies of hAng with PCNA in vitro , and their interaction was quantified by ITC, obtaining information on stoichiometry, enthalpy, entropy, and binding kinetics of the association. The hAng-PCNA association is strong, with a K d value of 126 nM...

Succinyl-CoA reductase (SucD) is an acylating aldehyde reductase that catalyzes the NADPH-dependent reduction of succinyl-CoA to succinic semialdehyde. The reaction sequence from succinate to crotonyl-CoA is of particular interest for several new-to-nature CO2 -fixation pathways, such as the crotonyl-CoA/ethylmalonyl-CoA/hydroxybutyryl-CoA (CETCH) cycle, in which SucD plays a key role. However, pathways like the CETCH cycle feature several CoA-ester intermediates, which could be potentially side substrates for this enzyme...

A major challenge in defining the pathophysiology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is to better understand virally encoded multifunctional proteins and their interactions with host factors. Among the many proteins encoded by the positive-sense, single-stranded RNA genome, nonstructural protein 1 (Nsp1) stands out due to its impact on several stages of the viral replication cycle. Nsp1 is the major virulence factor that inhibits mRNA translation. Nsp1 also promotes host mRNA cleavage to modulate host and viral protein expression and to suppress host immune functions...

5'-Methylthioadenosine nucleosidases (MTANs) catalyze the hydrolysis of 5'-substituted adenosines to form adenine and 5-substituted ribose. Escherichia coli MTAN ( Ec MTAN) and Helicobacter pylori MTAN ( Hp MTAN) form late and early transition states, respectively. Transition state analogues designed for the late transition state bind with fM to pM affinity to both classes of MTANs. Here, we compare the residence times (off-rates) with the equilibrium dissociation constants for Hp MTAN and Ec MTAN, using five 5'-substituted DADMe-ImmA transition state analogues...

Saccharomyces cerevisiae IA3 is a 68 amino acid peptide inhibitor of yeast proteinase A (YPRA) characterized as a random coil when in solution, folding into an N-terminal amphipathic alpha helix for residues 2-32 when bound to YPRA, with residues 33-68 unresolved in the crystal complex. Circular dichroism (CD) spectroscopy results show that amino acid substitutions that remove hydrogen-bonding interactions observed within the hydrophilic face of the N-terminal domain (NTD) of IA3 -YPRA crystal complex reduce the 2,2,2-trifluoroethanol (TFE)-induced helical transition in solution...

Noble gases have well-established biological effects, yet their molecular mechanisms remain poorly understood. Here, we investigated, both experimentally and computationally, the molecular modes of xenon (Xe) action in bacteriophage T4 lysozyme (T4L). By combining indirect gassing methods with a colorimetric lysozyme activity assay, a reversible, Xe-specific (20 ± 3)% inhibition effect was observed. Accelerated molecular dynamic simulations revealed that Xe exerts allosteric inhibition on the protein by expanding a C-terminal hydrophobic cavity...

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