System-wide identification and prioritization of enzyme substrates by thermal analysis

Despite the immense importance of enzyme–substrate reactions, there is a lack of general and unbiased tools for identifying and prioritizing substrate proteins that are modified by the enzyme on the structural level. Here we describe a high-throughput unbiased proteomics method called System-wide Identification and prioritization of Enzyme Substrates by Thermal Analysis (SIESTA). The approach assumes that the enzymatic post-translational modification of substrate proteins is likely to change their thermal stability. In our proof-of-concept studies, SIESTA successfully identifies several known and novel substrate candidates for selenoprotein thioredoxin reductase 1, protein kinase B (AKT1) and poly-(ADP-ribose) polymerase-10 systems. Wider application of SIESTA can enhance our understanding of the role of enzymes in homeostasis and disease, opening opportunities to investigate the effect of post-translational modifications on signal transduction and facilitate drug discovery.

SIESTA as a universal unbiased proteomics approach for identification and prioritization of enzyme substrates

This protocol describes the proteomics technique called System-wide Identification and prioritization of Enzyme Substrates by Thermal Analysis or SIESTA 1,2. SIESTA can be used for universal discovery of enzyme substrates that shift in thermal stability or solubility upon post-translational modification (PTM). Experimental design, proteomics sample preparation and data analysis are the key stages of this protocol. Data analysis can be performed using our SIESTA package hosted on GitHub 3. When performed with classical thermal proteome profiling (TPP), the protocol will take 5 days for sample preparation and 14 days of sample analysis by mass spectrometry (the current protocol). If our high-throughput version of TPP called Proteome Integral Solubility Alteration assay (PISA) 4 is used instead, the sample analysis time by mass spectrometry is reduced to 1-2 days for the same number of conditions.

Screening and Optimization of Protease Enzyme Produced by Strains of Alkalihalobacillus Sp. and Bacillus Sp.

Background : Proteases are the most important industrial enzymes with diverse applications in. Bacteria, such as Bacillus, commonly used to produce protease for industrial purposes. Proteases are commercially exploited in large-scale, especially in pharmaceutical, food, leather and detergent industries. Objective: The aim of this study was screening and optimization of protease enzyme activity produced by local bacteria. Method: In this research, the effect of incubation time, temperature and initial pH were investigated to improve the extracellular protease enzyme activity by two bacteria, named Bacillus subtilis strain DAR and Alkalihalobacillus hwajinpoensis strain 3NB. These two isolates have already been isolated and registered from Iran. Results: The results indicated that the optimum incubation time for protease activity in B. subtilis strain DAR is 36 h in contrast to 40 h in Alkalihalobacillus hwajinpoensis strain 3NB. The optimum incubation temperatures for enzyme activity for B. subtilis and Alkalihalobacillus hwajinpoensis is 50°C and 40°C, respectively. Optimum pH conditions for protease activity for both of the bacteria is 8. Conclusion: In current study, we investigated the optimum incubation time, pH and temperature for best protease activity. Further studies are recommended to improve protease activity through changing enzyme substrates.

Detection of Microbial Nitroreductase Activity by Monitoring Exogenous Volatile Organic Compound Production Using HS-SPME-GC-MS

Development of a rapid approach for universal microbial detection is required in the healthcare, food and environmental sectors to aid with medical intervention, food safety and environmental protection. This research investigates the use of enzymatic hydrolysis of a substrate by a microorganism to generate a volatile organic compound (VOC). One such enzyme activity that can be used in this context is nitroreductase as such activity is prevalent across a range of microorganisms. A study was developed to evaluate a panel of 51 microorganisms of clinical interest for their nitroreductase activity. Two enzyme substrates, nitrobenzene and 1-fluoro-2-nitrobenzene, were evaluated for this purpose with evolution, after incubation, of the VOCs aniline and 2-fluoroaniline, respectively. Detection of the VOCs was done using headspace-solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) with obtained limits of quantitation (LOQ) of 0.17 and 0.03 µg/mL for aniline and 2-fluoroaniline, respectively. The results indicated that both enzyme substrates were reduced by the same 84.3% of microorganisms producing the corresponding volatile anilines which were detected using HS-SPME-GC-MS. It was found that nitroreductase activity could be detected after 6–8 h of incubation for the selected pathogenic bacteria investigated. This approach shows promise as a rapid universal microbial detection system.

A Click-Flipped Enzyme Substrate Boosts the Performance of the Diagnostic Screening for Hunter Syndrome

We report on the unexpected finding that click modification of iduronyl azides results in a conformational flip of the pyranose ring, which led to the development of a new strategy for the design of superior enzyme substrates for the diagnostic assaying of iduronate-2-sulfatase (I2S), a lysosomal enzyme related to Hunter syndrome. Synthetic substrates are essential in testing newborns for metabolic disorders to enable early initiation of therapy. Our click-flipped iduronyl triazole showed a remarkably better performance with I2S than commonly used <i>O</i>-iduronates. We found that both <i>O</i>- and triazole-linked substrates are accepted by the enzyme, irrespective of their different conformations, but only the <i>O</i>-linked product inhibits the activity of I2S. Thus, in the long reaction times required for clinical assays, the triazole substrate substantially outperforms the <i>O</i>-iduronate. Applying our click-flipped substrate to assay I2S in dried blood spots sampled from affected patients and random newborns significantly increased the confidence in discriminating between these groups, clearly indicating the potential of the click-flip strategy to control the biomolecular function of carbohydrates.

A Click-Flipped Enzyme Substrate Boosts the Performance of the Diagnostic Screening for Hunter Syndrome

We report on the unexpected finding that click modification of iduronyl azides results in a conformational flip of the pyranose ring, which led to the development of a new strategy for the design of superior enzyme substrates for the diagnostic assaying of iduronate-2-sulfatase (I2S), a lysosomal enzyme related to Hunter syndrome. Synthetic substrates are essential in testing newborns for metabolic disorders to enable early initiation of therapy. Our click-flipped iduronyl triazole showed a remarkably better performance with I2S than commonly used <i>O</i>-iduronates. We found that both <i>O</i>- and triazole-linked substrates are accepted by the enzyme, irrespective of their different conformations, but only the <i>O</i>-linked product inhibits the activity of I2S. Thus, in the long reaction times required for clinical assays, the triazole substrate substantially outperforms the <i>O</i>-iduronate. Applying our click-flipped substrate to assay I2S in dried blood spots sampled from affected patients and random newborns significantly increased the confidence in discriminating between these groups, clearly indicating the potential of the click-flip strategy to control the biomolecular function of carbohydrates.

Modified Enzyme Substrates for the Detection of Bacteria: A Review

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.

Biological Sensing and Imaging Using Conjugated Polymers and Peptide Substrates

: Peptides have been widely applied as targeting elements or enzyme-substrates in biological sensing and imaging. Conjugated polymers (CPs) have emerged as a novel biosensing material and received considerable attention due to their excellent light absorption, strong fluorescence emission, as well as amplified quenching properties. In this review, we summarize the recent advances of using CPs and peptide substrates in biosensing and bioimaging. After a brief introduction of the advantages of CPs and peptide substrates, different sensing designs and mechanisms are discussed based on peptides’ structures and functions, including targeting recognition elements, enzyme-substrates, and cell-penetrating elements. Applications of CPs and peptides in fluorescent imaging and Raman imaging in living cells are subsequently reviewed.

Synthesis and Antimicrobial Activity of Phosphonopeptide Derivatives Incorporating Single and Dual Inhibitors

In diagnostic microbiology, culture media are widely used for detection of pathogenic bacteria. Such media employ various ingredients to optimize detection of specific pathogens such as chromogenic enzyme substrates and selective inhibitors to reduce the presence of commensal bacteria. Despite this, it is rarely possible to inhibit the growth of all commensal bacteria, and thus pathogens can be overgrown and remain undetected. One approach to attempt to remedy this is the use of “suicide substrates” that can target specific bacterial enzymes and selectively inhibit unwanted bacterial species. With the purpose of identifying novel selective inhibitors, six novel phosphonopeptide derivatives based on d/l-fosfalin and β-chloro-l-alanine were synthesized and tested on 19 different strains of clinically relevant bacteria. Several compounds show potential as useful selective agents that could be exploited in the recovery of several bacterial pathogens including Salmonella, Pseudomonas aeruginosa, and Listeria.