Queueing up for enzymatic processing: correlated signaling through coupled degradation

High‐throughput technologies have led to the generation of complex wiring diagrams as a post‐sequencing paradigm for depicting the interactions between vast and diverse cellular species. While these diagrams are useful for analyzing biological systems on a large scale, a detailed understanding of the molecular mechanisms that underlie the observed network connections is critical for the further development of systems and synthetic biology. Here, we use queueing theory to investigate how ‘waiting lines’ can lead to correlations between protein ‘customers’ that are coupled solely through a downstream set of enzymatic ‘servers’. Using the E. coli ClpXP degradation machine as a model processing system, we observe significant cross‐talk between two networks that are indirectly coupled through a common set of processors. We further illustrate the implications of enzymatic queueing using a synthetic biology application, in which two independent synthetic networks demonstrate synchronized behavior when common ClpXP machinery is overburdened. Our results demonstrate that such post‐translational processes can lead to dynamic connections in cellular networks and may provide a mechanistic understanding of existing but currently inexplicable links.     

Microdialysis coupled to online enzymatic assays

In vivo sampling of interstitial fluid by using microdialysis fibers has become a standard and accepted procedure. This sampling method is generally coupled to offline analysis of consecutive dialysate samples by high-performance liquid chromatography or capillary electrophoresis, but this combination is not the best approach for some applications, especially those which require high temporal resolution and rapid data collection. The purpose of this review is to provide information on enzyme-based online assays, i.e., continuous analysis of the dialysate as it emerges from the outlet of the sampling device. We have focused on methods developed specifically for the analysis of solutions perfused at a very slow flow rate, i.e., a feature of microdialysis and ultrafiltration techniques. These methods include flow enzyme-fluorescence assays, flow enzyme-amperometric assays, and sequential enzyme-amperometric detection. Each type of assay is discussed in terms of principle, applications, advantages, and limitations. We also comment on implantable biosensors, an obvious next step forward for in vivo monitoring of molecules in neuroscience.     

Measurement of oxidized glutathione by enzymatic recycling coupled to bioluminescent detection

A new method is described for the quantification of oxidized glutathione (GSSG) in tissues by enzymatic recycling coupled to NADPH bioluminescent detection. Tissue samples are treated with metaphosphoric acid. In a first step, after derivatization of GSH with 4-chloro-7-trifluoromethyl-1-methylquinolinium (CFQ), GSSG is recycled in the presence of dithionitrobenzoic acid (DTNB) and NADPH by glutathione reductase. In a second step, the GSSG-dependent NADPH consumption is measured by luminescence with NADPH:FMN oxidoreductase-bacterial luciferase. The coefficient of variation for GSSG measurements on repeated assays (n = 5) is 2 and 3% for standards and tissue samples, respectively. The sensitivity of this method is at the picomole level and is convenient for determination of GSSG physiological concentrations in tissues: GSSG levels measured in rat liver and kidney ranged from 76 to 215 and 52 to 170 nmol/g wet weight, respectively.     

A deep learning approach to evaluate the feasibility of enzymatic reactions generated by retrobiosynthesis

Retrobiosynthesis allows the designing of novel biosynthetic pathways for the production of chemicals and materials through metabolic engineering, but generates a large number of reactions beyond the experimental feasibility. Thus, an effective method that can reduce a large number of the initially predicted enzymatic reactions has been needed. Here, we present Deep learning-based Reaction Feasibility Checker (DeepRFC) to classify the feasibility of a given enzymatic reaction with high performance and speed. DeepRFC is designed to receive Simplified Molecular-Input Line-Entry System (SMILES) strings of a reactant pair, which is defined as a substrate and a product of a reaction, as an input, and evaluates whether the input reaction is feasible. A deep neural network is selected for DeepRFC as it leads to better classification performance than five other representative machine learning methods examined. For validation, the performance of DeepRFC is compared with another in-house reaction feasibility checker that uses the concept of reaction similarity. Finally, the use of DeepRFC is demonstrated for the retrobiosynthesis-based design of novel one-carbon assimilation pathways. DeepRFC will allow retrobiosynthesis to be more practical for metabolic engineering applications by efficiently screening a large number of retrobiosynthesis-derived enzymatic reactions. DeepRFC is freely available at https://bitbucket.org/kaistsystemsbiology/deeprfc .     

Reduced models of networks of coupled enzymatic reactions

The Michaelis-Menten equation has played a central role in our understanding of biochemical processes. It has long been understood how this equation approximates the dynamics of irreversible enzymatic reactions. However, a similar approximation in the case of networks, where the product of one reaction can act as an enzyme in another, has not been fully developed. Here we rigorously derive such an approximation in a class of coupled enzymatic networks where the individual interactions are of Michaelis-Menten type. We show that the sufficient conditions for the validity of the total quasi-steady state assumption (tQSSA), obtained in a single protein case by Borghans, de Boer and Segel can be extended to sufficient conditions for the validity of the tQSSA in a large class of enzymatic networks. Secondly, we derive reduced equations that approximate the network's dynamics and involve only protein concentrations. This significantly reduces the number of equations necessary to model such systems. We prove the validity of this approximation using geometric singular perturbation theory and results about matrix differentiation. The ideas used in deriving the approximating equations are quite general, and can be used to systematize other model reductions.     

Distinct antigen MHC class II complexes generated by separate processing pathways.

The peptide binding site of MHC class II molecules is open at both ends and, therefore, does not restrict the length of the bound ligand. Here we show that a partially folded protein antigen (*HEL) spontaneously formed SDS-unstable complexes with the purified MHC class II molecule I-Ak (Ak). These complexes were also detected on the surface of antigen-presenting cells (APCs) where they stimulated T cells. However, they rapidly disappeared after endocytosis. Intracellular processing of *HEL gave rise to SDS-stable, long-lived Ak complexes containing *HEL peptides and, unexpectedly, full-length *HEL. Both SDS-stable products were formed in low pH compartments and then transported to the plasma membrane. In contrast to *HEL peptides, the stable association of *HEL occurred in an alternative pathway that required mature class II molecules and did not involve HLA-DM or proteases. SDS-stable *HEL-Ak complexes were formed by a reaction of endosomal Ak with endocytosed *HEL, but not by direct conversion of SDS-unstable complexes derived from the plasma membrane. Our work establishes a fundamental difference between the two MHC class II loading pathways and for the first time demonstrates a full-length protein as a product of antigen processing.     

Degradation of cytokinins by maize cytokinin dehydrogenase is mediated by free radicals generated by enzymatic oxidation of natural benzoxazinones

Hydroxamic acid 2,4‐dihydroxy‐7‐methoxy‐1,4‐benzoxazin‐one (DIMBOA) was isolated from maize phloem sap as a compound enhancing the degradation of isopentenyl adenine by maize cytokinin dehydrogenase (CKX), after oxidative conversion by either laccase or peroxidase. Laccase and peroxidase catalyze oxidative cleavage of DIMBOA to 4‐nitrosoresorcinol‐1‐monomethyl ether (coniferron), which serves as a weak electron acceptor of CKX. The oxidation of DIMBOA and coniferron generates transitional free radicals that are used by CKX as effective electron acceptors. The function of free radicals in the CKX‐catalyzed reaction was also verified with a stable free radical of 2,2′‐azino‐bis‐3‐ethylbenzothiazoline‐6‐sulfonic acid. Application of exogenous cytokinin to maize seedlings resulted in an enhanced benzoxazinoid content in maize phloem sap. The results indicate a new function for DIMBOA in the metabolism of the cytokinin group of plant hormones.     

Preparation of 2-aminomuconate from 2-aminophenol by coupled enzymatic dioxygenation and dehydrogenation reactions

2-Aminomuconate is an intermediate in the oxidative metabolism of tryptophan in mammals. The compound is not commercially available, and studies of its metabolism have been prevented by the lack of a chemical synthesis and the instability of the molecule. We report here the formation of 2-aminomuconate from 2-aminophenol by the coupled action of 2-aminophenol 1,6-dioxygenase and 2-aminomuconic semialdehyde dehydrogenase from Pseudomonas pseudoalcaligenes JS45, and isolation of the product by anion exchange chromatography. The overall procedure was completed within 3 h with a yield of 62%. The availability of the dicarboxyl α-amino acid provides the basis for investigation of the physiological function of 2-aminomuconate in the neuropathologically significant oxidative metabolism of tryptophan. Received 23 February 1999/ Accepted in revised form 09 June 1999     

Fermentation of cellulosic hydrolysates obtained by enzymatic saccharification of sugarcane bagasse pretreated by hydrothermal processing

This work aims to evaluate the fermentability of cellulosic hydrolysates obtained by enzymatic saccharification of sugarcane bagasse pretreated by hydrothermal processing using Candida guilliermondii FTI 20037 yeast. The inoculum was obtained from yeast culture in a medium containing glucose as a carbon source supplemented with rice bran extract, CaCl₂·2H₂O and (NH₄)₂SO₄ in 50 mL Erlenmeyer flasks, containing 20 mL of medium, initial 5.5 pH under agitation of an orbital shaker (200 rpm) at 30°C for 24 h. The cellulosic hydrolysates, prior to being used as a fermentation medium, were autoclaved for 15 min at 0.5 atm and supplemented with the same nutrients employed for the inoculum, except the glucose, using the same conditions for the inoculum, but with a period of 48 h. Preliminary results showed the highest consumption of glucose (97%) for all the hydrolysates, at 28 h of fermentation. The highest concentration of ethanol (20.5 g/L) was found in the procedure of sugarcane bagasse pretreated by hydrothermal processing (195°C/10 min in 20 L reactor) and delignificated with NaOH 1.0% (w/v), 100°C, 1 h in 500 mL stainless steel ampoules immersed in an oil bath.     

A proteomic view of Bifidobacterium infantis generated by multi-dimensional chromatography coupled with tandem mass spectrometry

Bifidobacteria are Gram-positive prokaryotes that naturally colonize the human gut where they exert several health-promoting effects. The present paper reports the use of a strong cation exchange-reversed-phase-tandem mass spectrometry strategy to catalogue the most abundantly expressed proteins of a probiotic Bifidobacterium infantis strain. A global view of the B. infantis proteome was obtained. The bimodal representation of the proteins identified by mass spectrometry provides the first theoretical two-dimensional map of protein distribution for this organism. Among the 136 proteins identified by multidimensional protein identification technology (MudPIT) analysis, 118 showed the highest similarity with the translated sequences of B. longum genome, two proteins were similar to other Bifidobacterium species and the remaining 16 were similar to different genera. Specific biological activities have been assigned to 115 identified proteins, whereas 21 have been referred to the group of hypothetical proteins. The MudPIT approach allowed us to identify high mass and basic isoelectric point proteins that are generally challenging to visualize using the traditional two-dimensional electrophoresis technique. Redundancy in peptide and protein identification using the double chromatography technique was also evaluated.     

Deletion mutants of simian virus 40 generated by enzymatic excision of DNA segments from the viral genome

Deleted genomes of simian virus 40 have been constructed by enzymatic excision of specific segments of DNA from the genome of wild-type SV40². For this purpose, a restriction endonuclease from Hemophilus influenzae (endo R · HindIII) was used. This enzyme cleaves SV40 DNA into six fragments, which have cohesive termini. Partial digest products were separated by electrophoresis in agarose gel and subsequently cloned by plaque formation in the presence of complementing temperature-sensitive mutants of SV40. Individual deletion mutants generated in this way were mapped by analysis of DNA fragments produced by endo R · Hind digestion of their deleted genomes, and by heteroduplex mapping. Two types of deletions were found: (1) “excisional” deletions, in which the limits of the deleted segment corresponded to HindIII cleavage sites, and (2) “extended” deletions, in which the deleted segment extended beyond HindIII cleavage sites. Excisionally deleted genomes presumably arose by cyclization of a linear fragment via cohesive termini generated by endo R · HindIII whereas genomes with extended deletions probably were generated by intramolecular recombination near the ends of linear fragments. Of the nine mutants analyzed, two had deletions in the “early” region of the SV40 genome, six had deletions in the “late” region, and one had a deletion that spanned both regions.     

Secreted alpha amidating enzymes are generated by specific posttranslational processing of precursors containing transmembrane domains

The biosynthesis and secretion of alpha amidating enzymes from CA-77 cells has been investigated to determine the relationship among the various forms of alpha amidating enzyme seen after purification of alpha amidating enzyme activity from conditioned cell culture media. Initially 2 proteins of 104 kD and 94 kD are synthesized. With time the 104 kD precursor is processed to 41 kD and 43 kD, and the 94 kD precursor is processed to 75 kD. The 41 kD, 43 kD, and 75 kD proteins are secreted into the medium as functional enzymes. In comparing these data with known cDNA sequence for alpha amidating enzyme we conclude that the 104 kD and 94 kD precursors are membrane bound proteins which are posttranslationally processed to generate secreted alpha amidating enzyme.     

Enhanced rates of enzymatic saccharification and catalytic synthesis of biofuel substrates in gelatinized cellulose generated by trifluoroacetic acid

Background

The crystallinity of cellulose is a principal factor limiting the efficient hydrolysis of biomass to fermentable sugars or direct catalytic conversion to biofuel components. We evaluated the impact of TFA-induced gelatinization of crystalline cellulose on enhancement of enzymatic digestion and catalytic conversion to biofuel substrates.

Results

Low-temperature swelling of cotton linter cellulose in TFA at subzero temperatures followed by gentle heating to 55 °C dissolves the microfibril structure and forms composites of crystalline and amorphous gels upon addition of ethanol. The extent of gelatinization of crystalline cellulose was determined by reduction of birefringence in darkfield microscopy, loss of X-ray diffractability, and loss of resistance to acid hydrolysis. Upon freeze-drying, an additional degree of crystallinity returned as mostly cellulose II. Both enzymatic digestion with a commercial cellulase cocktail and maleic acid/AlCl₃-catalyzed conversion to 5-hydroxymethylfurfural and levulinic acid were markedly enhanced with the low-temperature swollen cellulose. Only small improvements in rates and extent of hydrolysis and catalytic conversion were achieved upon heating to fully dissolve cellulose.

Conclusions

Low-temperature swelling of cellulose in TFA substantially reduces recalcitrance of crystalline cellulose to both enzymatic digestion and catalytic conversion. In a closed system to prevent loss of fluorohydrocarbons, the relative ease of recovery and regeneration of TFA by distillation makes it a potentially useful agent in large-scale deconstruction of biomass, not only for enzymatic depolymerization but also for enhancing rates of catalytic conversion to biofuel components and useful bio-products.

     

A coupled assay measuring Mycobacterium tuberculosis antigen 85C enzymatic activity

The prevalence of drug-resistant strains of Mycobacterium tuberculosis (M. tb) emphasizes the need for new antitubercular drugs. An essential component of the drug discovery process is the development of tools to rapidly screen potential drug libraries against important biological targets. Similarly to well-documented M. tb targets, the antigen 85 (Ag85) enzymes are involved in the maintenance of the mycobacterial cell wall. The products synthesized by these mycolyltransferases are the cell wall components most responsible for the reduced permeability of drugs into the bacterial cell, thereby linking Ag85 activity directly with drug resistance. This article presents the development of a high-throughput colorimetric assay suitable for direct monitoring of the enzymatic activity. The assay uses a synthetic substrate containing three chemical moieties: an octanoyl fatty acid, β-d-glucose, and p-nitrophenyl. In the context of the assay, Ag85 catalyzes the removal of the fatty acid and releases p-nitrophenyl-β-d-glucoside. The glucoside is hydrolyzed by β-glucosidase to release the p-nitrophenolate chromophore. With this assay, the K_M and k_cat values of Ag85C were determined to be 0.047 ± 0.008 mM and 0.062 s⁻¹, respectively. In addition, the assay exhibits a Z′ value of 0.81 ± 0.06, indicating its suitability for high-throughput screening applications and drug development.     

Plasma processing of spent nuclear fuel by two-frequency ion cyclotron resonance heating

A previously developed method for analyzing the plasma processing of spent nuclear fuel is generalized to a plasma containing multicharged fuel ions. In such a plasma, ion cyclotron resonance heating of nuclear ash ions should be carried out in two monochromatic RF fields of different frequencies, provided that the fraction of ξ multicharged ions is small, ξ ≤ 0.1, a condition that substantially restricts the productivity of systems for processing spent nuclear fuel. Ways of overcoming this difficulty are discussed.     

Three different coupled enzymatic systems for in situ regeneration of NADPH

Three different coupled enzymatic systems used in the reduction of sulcatone by alcohol dehydrogenase from Thermoanaerobium brockii (TBADH), were kinetically compared. The first one involved the use of TBADH for both the principal and recycling reactions and 2-propanol 20%, v/v as the recycling substrate. The other two were based on the use of a different enzyme, glucose- or glucose-6-phosphate dehydrogenases, for in situ regeneration of NADPH. The coupled-substrate approach achieved 100% of conversion against 84% of the other two systems.