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The recruitment and evolutionary optimization of promiscuous enzymes is key to the rapid adaptation of organisms to changing environments. Our understanding of the precise mechanisms underlying enzyme repurposing is, however, limited: What are the active-site features that enable the molecular recognition of multiple substrates with contrasting catalytic requirements? To gain insights into the molecular determinants of adaptation in promiscuous enzymes, we performed the laboratory evolution of an arylsulfatase to improve its initially weak phenylphosphonate hydrolase activity. The evolutionary trajectory led to a 100,000-fold enhancement of phenylphosphonate hydrolysis, while the native sulfate and promiscuous phosphate mono- and diester hydrolyses were only marginally affected (≤50-fold). Structural, kinetic, and in silico characterizations of the evolutionary intermediates revealed that two key mutations, T50A and M72V, locally reshaped the active site, improving access to the catalytic machinery for the phosphonate. Measured transition state (TS) charge changes along the trajectory suggest the creation of a new Michaelis complex (E•S, enzyme–substrate), with enhanced leaving group stabilization in the TS for the promiscuous phosphonate (β leaving group from −1.08 to −0.42). Rather than altering the catalytic machinery, evolutionary repurposing was achieved by fine-tuning the molecular recognition of the phosphonate in the Michaelis complex, and by extension, also in the TS. This molecular scenario constitutes a mechanistic alternative to adaptation solely based on enzyme flexibility and conformational selection. Instead, rapid functional transitions between distinct chemical reactions rely on the high reactivity of permissive active-site architectures that allow multiple substrate binding modes.

In this study, the concentrations of nine typical antibiotics, including sulfadiazine (SD), sulfamerazine (SMR), sulfamethazine (SM2), sulfamethoxazole (SMZ), ofloxacin (OFX), ciprofloxacin (CIP), trimethoprim (TMP), oxytetracycline (OTC), and tetracycline hydrochloride (TC), were detected in the Yitong River by solid-phase extraction high-performance liquid chromatography. The concentrations of the antibiotics were analyzed. Additionally, an improved immobilized substrate enzyme substrate method (DST-enzyme substrate method) was developed and used to evaluate the antibiotic resistance of coliform bacteria to OFX, CIP, enrofloxacin (ENR), TC, sulfisoxazole (SOX), and TMP in the Yitong River. The results showed that the concentrations of the nine antibiotics ranged from nd (not detected) to 1.361 μg/L. The detection rate and concentration of OFX were the highest, followed by CIP, and the detection rate and concentration of SM2 and OTC were the lowest. The detection rate and concentrations of antibiotics were higher in August and November than those in May. The antibiotics were mainly distributed in the livestock sewage discharge and suburban domestic sewage discharge areas. Moreover, the drug resistance of total coliform bacteria to fluoroquinolones, sulfonamides, tetracyclines, and TMP varied with season.

An analysis of the enzymatic hydrolysis of wheat starch was performed. The gelatinization stage was carried out between 90-95°C for 15min. In the liquefaction stage, a commercial α-amylase was used with an enzyme-substrate ratio (E/S ratio) 0.036%w/w at 60°C and pH 5.8 for 4h. In the saccharification stage, a commercial amyloglucosidase was used with an E/S ratio of 0.11% w/w at 60°C and pH 4.3 for 6h. A second hydrolysis was evaluated using a E/S ratio of  0.18%w/w in the saccharification stage. Two methods of enzymatic deactivation, boiling temperatures and pH were evaluated. Inhibitory effects were studied by adding 180g/L of glucose to the process. It is concluded that increases in the E/S ratio decrease reaction times but reaches similar concentrations than lower ratios, the most efficient enzymatic deactivation method is pH. In the inhibition tests, it was determined that there are no glucose inhibitory effects.

The Michaelis—Menten equation provides a hundred-year-old prediction by which any increase in the rate of substrate unbinding will decrease the rate of enzymatic turnover. Surprisingly, this prediction was never tested experimentally nor was it scrutinized using modern theoretical tools. Here we show that unbinding may also speed up enzymatic turnover—turning a spotlight to the fact that its actual role in enzymatic catalysis remains to be determined experimentally. Analytically constructing the unbinding phase space, we identify four distinct categories of unbinding: inhibitory, excitatory, superexcitatory, and restorative. A transition in which the effect of unbinding changes from inhibitory to excitatory as substrate concentrations increase, and an overlooked tradeoff between the speed and efficiency of enzymatic reactions, are naturally unveiled as a result. The theory presented herein motivates, and allows the interpretation of, groundbreaking experiments in which existing single-molecule manipulation techniques will be adapted for the purpose of measuring enzymatic turnover under a controlled variation of unbinding rates. As we hereby show, these experiments will not only shed first light on the role of unbinding but will also allow one to determine the time distribution required for the completion of the catalytic step in isolation from the rest of the enzymatic turnover cycle.

It is still problematic to use enzyme activities as indicators of soil functions because: (1) enzyme assays determine potential and not real enzyme activities; (2) the meaning of measured enzyme activities is not known; (3) the assumption that a single enzyme activity is an indicator of nutrient dynamics in soil neglects that the many enzyme activities are involved in such dynamic processes; (4) spatio-temporal variations in natural environments are not always considered when measuring enzyme activities; and (5) many direct and indirect effects make difficult the interpretation of the response of the enzyme activity to perturbations, changes in the soil management, changes in the plant cover of soil, etc. This is the first review discussing the links between enzyme-encoding genes and the relative enzyme activity of soil. By combining measurements of enzyme activity in soil with expression (transcriptomics and proteomics) of genes, encoding the relative enzymes may contribute to understanding the mode and timing of microbial communities’ responses to substrate availability and persistence and stabilization of enzymes in the soil.

BackgroundThe top-down analysis of nitrate influx isotherms through the Enzyme-Substrate interpretation has not withstood recent molecular and histochemical analyses of nitrate transporters. Indeed, at least four families of nitrate transporters operating at both high and/or low external nitrate concentrations, and which are located in series and/or parallel in the different cellular layers of the mature root, are involved in nitrate uptake. Accordingly, the top-down analysis of the root catalytic structure for ion transport from the Enzyme-Substrate interpretation of nitrate influx isotherms is inadequate. Moreover, the use of the Enzyme-Substrate velocity equation as a single reference in agronomic models is not suitable in its formalism to account for variations in N uptake under fluctuating environmental conditions. Therefore, a conceptual paradigm shift is required to improve the mechanistic modelling of N uptake in agronomic models.ScopeAn alternative formalism, the Flow-Force theory, was proposed in the 1970s to describe ion isotherms based upon biophysical ‘flows and forces’ relationships of non-equilibrium thermodynamics. This interpretation describes, with macroscopic parameters, the patterns of N uptake provided by a biological system such as roots. In contrast to the Enzyme-Substrate interpretation, this approach does not claim to represent molecular characteristics. Here it is shown that it is possible to combine the Flow-Force formalism with polynomial responses of nitrate influx rate induced by climatic and in planta factors in relation to nitrate availability.ConclusionsApplication of the Flow-Force formalism allows nitrate uptake to be modelled in a more realistic manner, and allows scaling-up in time and space of the regulation of nitrate uptake across the plant growth cycle.

Ubiquitin carboxy-terminal hydrolase L1 (UCHL1) is a Parkinson disease-associated, putative cysteine protease found abundantly and selectively expressed in neurons. The crystal structure of apo UCHL1 showed that the active-site residues are not aligned in a canonical form, with the nucleophilic cysteine being 7.7 Å from the general base histidine, an arrangement consistent with an inactive form of the enzyme. Here we report the crystal structures of the wild type and two Parkinson disease-associated variants of the enzyme, S18Y and I93M, bound to a ubiquitin-based suicide substrate, ubiquitin vinyl methyl ester. These structures reveal that ubiquitin vinyl methyl ester binds primarily at two sites on the enzyme, with its carboxy terminus at the active site and with its amino-terminal β-hairpin at the distal site--a surface-exposed hydrophobic crevice 17 Å away from the active site. Binding at the distal site initiates a cascade of side-chain movements in the enzyme that starts at a highly conserved, surface-exposed phenylalanine and is relayed to the active site resulting in the reorientation and proximal placement of the general base within 4 Å of the catalytic cysteine, an arrangement found in productive cysteine proteases. Mutation of the distal-site, surface-exposed phenylalanine to alanine reduces ubiquitin binding and severely impairs the catalytic activity of the enzyme. These results suggest that the activity of UCHL1 may be regulated by its own substrate.

The recently discovered lytic polysaccharide monooxygenases (LPMOs) are known to carry out oxidative cleavage of glycoside bonds in chitin and cellulose, thus boosting the activity of well-known hydrolytic depolymerizing enzymes. Because biomass-degrading microorganisms tend to produce a plethora of LPMOs, and considering the complexity and copolymeric nature of the plant cell wall, it has been speculated that some LPMOs may act on other substrates, in particular the hemicelluloses that tether to cellulose microfibrils. We demonstrate that an LPMO from Neurospora crassa , Nc LPMO9C, indeed degrades various hemicelluloses, in particular xyloglucan. This activity was discovered using a glycan microarray-based screening method for detection of substrate specificities of carbohydrate-active enzymes, and further explored using defined oligomeric hemicelluloses, isolated polymeric hemicelluloses and cell walls. Products generated by Nc LPMO9C were analyzed using high performance anion exchange chromatography and multidimensional mass spectrometry. We show that Nc LPMO9C generates oxidized products from a variety of substrates and that its product profile differs from those of hydrolytic enzymes acting on the same substrates. The enzyme particularly acts on the glucose backbone of xyloglucan, accepting various substitutions (xylose, galactose) in almost all positions. Because the attachment of xyloglucan to cellulose hampers depolymerization of the latter, it is possible that the beneficial effect of the LPMOs that are present in current commercial cellulase mixtures in part is due to hitherto undetected LPMO activities on recalcitrant hemicellulose structures.

The ability to detect, identify and quantify bacteria is crucial in clinical diagnostics, environmental testing, food security settings and in microbiology research. Recently, the threat of multidrug-resistant bacterial pathogens pushed the global scientific community to develop fast, reliable, specific and affordable methods to detect bacterial species. The use of synthetically modified enzyme substrates is a convenient approach to detect bacteria in a specific, economic and rapid manner. The method is based on the use of specific enzyme substrates for a given bacterial marker enzyme, conjugated to a signalogenic moiety. Following enzymatic reaction, the signalophor is released from the synthetic substrate, generating a specific and measurable signal. Several types of signalophors have been described and are defined by the type of signal they generate, such as chromogenic, fluorogenic, luminogenic, electrogenic and redox. Signalophors are further subdivided into groups based on their solubility in water, which is key in defining their application on solid or liquid media for bacterial culturing. This comprehensive review describes synthetic enzyme substrates and their applications for bacterial detection, showing their mechanism of action and their synthetic routes.

Enzymes are thought to have evolved highly specific catalytic activities from promiscuous ancestral proteins. By analyzing a genome-scale model of Escherichia coli metabolism, we found that 37% of its enzymes act on a variety of substrates and catalyze 65% of the known metabolic reactions. However, it is not apparent why these generalist enzymes remain. Here, we show that there are marked differences between generalist enzymes anf specialist enzymes, known to catalyze a single chemical reaction on one particular substrate in vivo. Specialist enzymes (i) are frequently essential, (ii) maintain higher metabolic flux, and (iii) require more regulation of enzyme activity to control metabolic flux in dynamic environments than do generalist enzymes. Furthermore, these properties are conserved in Archaea and Eukarya. Thus, the metabolic network context and environmental conditions influence enzyme evolution toward high specificity.