It is a challenge for the traditional affinity methods to capture transient interactions of enzyme-post-translational modification (PTM) substrates in vivo. Herein we presented a strategy termed proximity labeling-based orthogonal trap approach (ProLORT), relying upon APEX2-catalysed proximity labeling and an orthogonal trap pipeline as well as quantitative proteomics to directly investigate the transient interactome of enzyme-PTM substrates in living cells. As a proof of concept, ProLORT allows for robust evaluation of a known HDAC8 substrate, histone H3K9ac. By leveraging this approach, we identified numerous of putative acetylated proteins targeted by HDAC8, and further confirmed CTTN as a bona fide substrate in vivo. Next, we demonstrated that HDAC8 facilitates cell motility via deacetylation of CTTN at lysine 144 that attenuates its interaction with F-actin, expanding the underlying regulatory mechanisms of HDAC8. We developed a general strategy to profile the transient enzyme-substrate interactions mediated by PTMs, providing a powerful tool for identifying the spatiotemporal PTM-network regulated by enzymes in living cells.

Aminoacyl-tRNA synthetases are an indispensable component of ribosomal protein translational machinery and Plasmodium Tyrosyl-tRNA synthetase (PfTyrRS) is a validated drug target. This manuscript illustrates the dynamic conformational landscape of PfTyrRS in the context of substrate binding. Molecular dynamics simulations of PfTyrRS in the presence and absence of ligand show conformational heterogeneity for both the protein and the bound ligand. Diverse conformations for the evolutionarily conserved ATP binding motif (KMSKS) have been observed in both apo- and holo PfTyrRS. Further, the presented attributes of the tyrosyl-adenylate conformational sub-states in situ along with their implications on the strength of intermolecular interactions would be a pertinent benchmark for molecular design studies. In addition, an analysis of the ligand hydration pattern foregrounds the structurally conserved water-mediated inter-molecular interactions. The quantitative assessment of the conformational landscape, based on the fluctuations of the distance between the ligand binding pockets, of apo-PfTyrRS and holo-PfTyrRS highlights the nature of diversity in conformational sampling for the two cases. Evidently, the holo-PfTyrRS adopts a rather compact conformation compared to the apo-PfTyrRS. An intriguing asymmetry in the dynamics of the two monomers is contextualized with the functional asymmetry of the symmetrically dimeric PfTyrRS. Importantly, the network of non-bonded contacts in the apo- and holo- simulated ensembles has been analyzed. The graph-theoretic analysis-based novel insights concerning the nature of information flow as a function of ligation state would prove valuable in understanding PfTyrRS functions. The results presented here contend that understanding allostery in PfTyrRS is essential to astutely design structure-based inhibitors.

Enzyme catalysis is omnipresent in the cell. The mechanisms by which highly evolved protein folds enable rapid and specific chemical transformation of substrates belong to the marvels of structural biology. Targeting of enzymes with inhibitors has immediate application in drug discovery, from chemotherapeutics over antibiotics to antivirals. NMR spectroscopy combines multiple assets for the investigation of enzyme function. The non-invasive technique can probe enzyme structure and dynamics and map interactions with substrates, cofactors and inhibitors at the atomic level. With experiments performed at close to native conditions, catalytic transformations can be monitored in real time, giving access to kinetic parameters. The power of NMR in the solid state, in contrast with solution, lies in the absence of fundamental size limitations, which is crucial for enzymes that are either membrane-embedded or assemble into large soluble complexes exceeding hundreds of kilodaltons in molecular weight. Here we review recent progress in solid-state NMR methodology, which has taken big leaps in the past years due to steady improvements in hardware design, notably magic angle spinning, and connect it to parallel biochemical advances that enable isotope labelling of increasingly complex enzymes. We first discuss general concepts and requirements of the method and then highlight the state-of-the-art in sample preparation, structure determination, dynamics and interaction studies. We focus on examples where solid-state NMR has been instrumental in elucidating enzyme mechanism, alone or in integrative studies.

Cytochrome P450 1A1 (CYP1A1) has served as a known metabolic enzyme that mediates the carcinogenesis of polycyclic aromatic hydrocarbons (PAHs). However, the structural mechanism involved in the metabolic capacity remains unclear. In this study, thirty-three calculated properties representing the physicochemical and electronic properties of PAH and PAH-CYP1A1 interactions were utilized to identify the key structural properties that affect metabolic processes, including binding ability, metabolic clearance, and mutagenicity, using a quantitative structure-activity relationship (QSAR) strategy combined with docking methods, QM/MM calculations and ab initio calculations. van der Waals interactions (glide vdw) appeared to be important for PAH binding to CYP1A1 and were mainly affected by the molecular weight and hydrophobic structures of PAHs. Interaction features between PAHs and heme, including the distance between iron and carbons of PAHs (Fe_Cmin) and heme vdw, coordinately influence the metabolic clearance of PAHs. Furthermore, the electronic properties (ESP neg variance) appeared to be critical for the mutagenicity of PAHs by CYP1A1 through influencing epoxide metabolite formation. The QSAR models with these key properties provide a new perspective on the structural mechanism of PAH metabolism and provide a useful in silico tool for screening, classifying and predicting PAHs for their metabolism-related toxicities and risk assessment in the environment.

Recycling biomass is indispensable these days not only because fossil energy sources are gradually depleted, but also because pollution of the environment, caused by the increasing use of energy, must be reduced. This article intends to overview the results of plant biomass processing methods that are currently in use. Our aim was also to review published methods that are not currently in use. It is intended to explore the possibilities of new methods and enzymes to be used in biomass recycling. The results of this overview are perplexing in almost every area. Advances have been made in the pre-treatment of biomass and in the diversity and applications of the enzymes utilized. Based on molecular modeling, very little progress has been made in the modification of existing enzymes for altered function and adaptation for the environmental conditions during the processing of biomass. There are hardly any publications in which molecular modeling techniques are used to improve enzyme function and to adapt enzymes to various environmental conditions. Our view is that using modern computational, biochemical, and biotechnological methods would enable the purposeful design of enzymes that are more efficient and suitable for biomass processing.

Despite the enormous number of therapeutic advances in medicine, nowadays many diseases are still incurable, mainly due to the lack of knowledge of the pathological biochemical pathways triggering those diseases. For this reason, it is compulsory for the scientific community to investigate and unveil the biomolecular mechanisms responsible for the development of those diseases, such as Alzheimer\'s disease and diabetes, which are widespread all over the world. In this scenario, it is of paramount importance to develop new analytical techniques and experimental procedures that are capable to make the above-mentioned investigations feasible. These new methods should allow easy performable analysis carried out in a label-free environment, in order to give reliable answers to specific biochemical questions. A recent paper published on Bioscience Reports by Ivancic et al. (https://doi.org/10.1042/BSR20181416) proposes a new analytical technique capable to reveal some mechanistic insights into the regulation of insulin-degrading enzyme (IDE), a protein involved in the above-mentioned diseases. IDE is a multifaceted enzyme having different and not well-defined roles in the cell, but it is primarily a proteolytic enzyme capable to degrade several different amyloidogenic substrates involved in different diseases. Moreover, many molecules are responsible for IDE activity modulation so that understanding how IDE activity is regulated represents a very challenging analytical task. The new analytical approach proposed by Ivancic et al. reports on the possibility to study IDE activity in an unbiased and label-free manner, representing a valid alternative assay for the investigation of any proteases degradative activity.

Compelling evidence suggests a crucial role of amyloid beta peptides (Aβ(1-40/42)) in the etiology of Alzheimer\'s disease (AD). The N-terminal truncation of Aβ(1-40/42) and their modification, e.g. by glutaminyl cyclase (QC), is expected to enhance the amyloid toxicity. In this work, the MALDI-TOF mass spectrometry application proved N-terminal cleavage of Aβ(1-40/42) by purified dipeptidyl peptidase IV (DPPIV) in vitro observed earlier. The subsequent transformation of resulted Aβ(3-40/42) to pE-Aβ(3-40/42) in QC catalyzed glutamate cyclization was manifested. Hence, consecutive conversion of Aβ(1-40/42) by DPPIV and QC can be assumed as a potential mechanism of formation of non-degrading pyroglutamated pE-Aβ(3-40/42), which might accumulate and contribute to AD progression. The in vitro acceleration of Aβ(1-40) aggregation in the simultaneous presence of DPPIV and QC was shown also.

The enzymatic hydrolysis of cellulose is a thermodynamically challenging catalytic process that is influenced by both substrate-related and enzyme-related factors. In this study, a proteolysis approach was applied to recover and clean the partially converted cellulose at the different stages of enzymatic hydrolysis to monitor the hydrolysis rate as a function of substrate reactivity/accessibility and investigate surface characteristics of the partially converted cellulose. Enzyme-substrate interactions between individual key cellulase components from wild-type Trichoderma reesei and partially converted cellulose were followed and correlated to the enzyme adsorption capacity and dynamic sugar release. Results suggest that cellobiohydrolase CBH1 (Cel7A) and endoglucanases EG2 (Cel5A) adsorption capacities decreased as cellulose was progressively hydrolyzed, likely due to the \"depletion\" of binding sites. Furthermore, the degree of synergism between CBH1 and EG2 varied depending on the enzyme loading and the substrates. The results provide a better understanding of the relationship between dynamic change of substrate features and the functionality of various cellulase components during enzymatic hydrolysis. Biotechnol. Bioeng. 2017;114: 503-515. © 2016 Wiley Periodicals, Inc.

Glycoside hydrolase (GH) family 65 contains phosphorylases acting on maltose (Glc-α1,4-Glc), kojibiose (Glc-α1,2-Glc), trehalose (Glc-α1,α1,-Glc), and nigerose (Glc-α1,3-Glc). These phosphorylases can efficiently catalyze the reverse reactions with high specificities, and thus can be applied to the practical synthesis of α-glucosyl oligosaccharides. Here, we determined the crystal structures of kojibiose phosphorylase from Caldicellulosiruptor saccharolyticus in complex with glucose and phosphate and in complex with kojibiose and sulfate, providing the first structural insights into the substrate recognition of a glycoside hydrolase family 65 enzyme. The loop 3 region comprising the active site of kojibiose phosphorylase is significantly longer than the active sites of other enzymes, and three residues around this loop, Trp391, Glu392, and Thr417, recognize kojibiose. Various mutants mimicking the residue conservation patterns of other phosphorylases were constructed by mutation at these three residues. Activity measurements of the mutants against four substrates indicated that Trp391 and Glu392, especially the latter, are required for the kojibiose activity.