Advances In Enzymology Research Articles

AARS urzymes: Experimental biochemistry to map genetic coding

AARS urzymes: Experimental biochemistry to map genetic coding Dr Charlie Carter from the University of North Carolina at Chapel Hill explores how advances in enzymology and phylogenetics enable biochemical measurements that could map the ancestral development of genetic coding. Chemical reactions in the cell degrade energy-rich food molecules and rearrange their chemical bonds into other molecules necessary to construct and sustain living things far from chemical equilibrium. Those reactions occur spontaneously at rates that span an extraordinary range, the fastest going up to 1025 times faster than the slowest. (1) All these reactions must be accelerated by different amounts to maintain adequate concentrations of chemical components in the cell. Enzymes perform that differential acceleration exquisitely well.

Peculiarities of meroterpenoids and their bioproduction.

Meroterpenoids are a class of terpenoid-containing hybrid natural products with impressive structural architectures and remarkable pharmacological activities. Remarkable advances in enzymology and synthetic biology have greatly contributed to the elucidation of the molecular basis for their biosynthesis. Here, we review structurally unique meroterpenoids catalyzed by novel enzymes and unusual enzymatic reactions over the period of last 5 years. We also discuss recent progress on the biomimetic synthesis of chrome meroterpenoids and synthetic biology-driven biomanufacturing of tropolone sesquiterpenoids, merochlorins, and plant-derived meroterpenoid cannabinoids. In particular, we focus on the novel enzymes involved in the biosynthesis of polyketide-terpenoids, nonribosomal peptide-terpenoids, terpenoid alkaloids, and meroterpenoid with unique structures. The biological activities of these meroterpenoids are also discussed. The information reviewed here might provide useful clues and lay the foundation for developing new meroterpenoid-derived drugs. KEY POINTS: • Meroterpenoids possess intriguing structural features and relevant biological activities. • Novel enzymes are involved in the biosynthesis of meroterpenoids with unique structures. • Biomimetic synthesis and synthetic biology enable the construction and manufacturing of complex meroterpenoids.

Characteristics of the Proteolytic Enzymes Produced by Lactic Acid Bacteria.

Over the past several decades, we have observed a very rapid development in the biotechnological use of lactic acid bacteria (LAB) in various branches of the food industry. All such areas of activity of these bacteria are very important and promise enormous economic and industrial successes. LAB are a numerous group of microorganisms that have the ability to ferment sugars into lactic acid and to produce proteolytic enzymes. LAB proteolytic enzymes play an important role in supplying cells with the nitrogen compounds necessary for their growth. Their nutritional requirements in this regard are very high. Lactic acid bacteria require many free amino acids to grow. The available amount of such compounds in the natural environment is usually small, hence the main function of these enzymes is the hydrolysis of proteins to components absorbed by bacterial cells. Enzymes are synthesized inside bacterial cells and are mostly secreted outside the cell. This type of proteinase remains linked to the cell wall structure by covalent bonds. Thanks to advances in enzymology, it is possible to obtain and design new enzymes and their preparations that can be widely used in various biotechnological processes. This article characterizes the proteolytic activity, describes LAB nitrogen metabolism and details the characteristics of the peptide transport system. Potential applications of proteolytic enzymes in many industries are also presented, including the food industry.

Transglutaminaz Enziminin Deve Sütünden Üretilen Yoğurdun Bazı Özellikleri Üzerine Etkisi

Bu çalışmada, deve sütünden yoğurt üretiminde mikrobiyel transglutaminaz (MTGaz) enzimi kullanılmıştır. Yapılan çalışmalarda deve sütünün lizozim, laktoperoksidaz, laktoferrin ve immunoglobulinler gibi antimikrobiyal maddelerce zengin olmasının bu sütten yoğurt üretimini engellediği ve yoğurt pıhtısının oluşmadığı rapor edilmiştir. Enzimolojideki gelişmelerle birlikte proteinlerin fonksiyonel özelliklerini ve besin değerlerini geliştirmek için enzimatik modifikasyonların kullanılması ile deve sütünden yoğurt üretiminde MTGaz enzimi kullanımının etkili olabileceği ileri sürülmüştür. Bu amaçla çalışmada deve sütünün protein oranı %6,2 oranında sodyum kazeinat, serum proteini konsantratı ve misellar kazein tozu ilavesiyle artırılmıştır. MTGaz enzimi 3 U ve 6 U konsantrasyonlarında kullanılmış ve enzim deve sütüne starter kültürüyle aynı zamanda ilave edilerek fermentasyona bırakılmıştır. Fermentasyon süresince her saatte viskozite, pH ve titrasyon asitliği (% laktik asit olarak) analizleri yapılmıştır. Viskozite artışı MTGaz enzimi ilavesiyle meydana gelen çapraz bağlanma sonucunda gerçekleşmiş olup oluşan çapraz bağlar SDS-PAGE ile protein fraksiyonlarının monomer band yoğunluğunun azalması üzerinden tespit edilmiştir. MTGaz enzim konsantrasyonu ne kadar yüksek olursa amino asitler arasındaki çapraz bağların da o kadar fazla ve relatif viskozitenin de o kadar yüksek olduğu saptanmıştır. Bunlara ilave olarak deve sütüne ilave edilen yoğurt bakterilerinin çoğalıp çoğalmadığı ve MTGaz enziminden etkilenip etkilenmediğini araştırmak için yoğurt bakterilerinin sayısı hem M17 agarda hem de MRS agarda belirlenmiştir. Denemeler sonucunda MTGaz enzim ilavesinin bakteri sayısı üzerine baskılayıcı etkisinin bulunmadığı saptanmıştır.

Analysis of Methylation, Acetylation, and Other Modifications in Bacterial Ribosomal Proteins.

A wide variety of posttranslational modifications of expressed proteins are known to occur in living organisms (Krishna R, Wold F. Post-translational modification of proteins. In: Meister A (ed) Advances in enzymology and related areas of molecular biology. Wiley, New York, 1993, pp 265-296). Although their presence in an organism cannot be predicted from the genome, these modifications can play critical roles in protein structure and function. The identification of posttranslational modifications is critical to our understanding of the functions of proteins involved in important biological pathways and mass spectrometry offers a fast, accurate method for observing them. A combined top-down/bottom-up approach can be used for identification and localization of posttranslational modifications of ribosomal proteins. This chapter describes procedures for analyzing Escherichia coli ribosomal proteins and their modifications by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. It also covers the analysis of gram-negative Caulobacter crescentus and gram-positive Bacillus subtilis ribosomal proteins by electrospray quadrupole time-of-flight (ESI-QTOF) mass spectrometry. Confirmation of the occurrence and localization of PTMs is obtained through mass spectrometric analysis of tryptic peptides.

Nitroreductase gene-directed enzyme prodrug therapy: insights and advances toward clinical utility.

This review examines the vast catalytic and therapeutic potential offered by typeI (i.e. oxygen-insensitive) nitroreductase enzymes in partnership with nitroaromatic prodrugs, with particular focus on gene-directed enzyme prodrug therapy (GDEPT; a form of cancer gene therapy). Important first indications of this potential were demonstrated over 20years ago, for the enzyme-prodrug pairing of Escherichia coli NfsB and CB1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide]. However, it has become apparent that both the enzyme and the prodrug in this prototypical pairing have limitations that have impeded their clinical progression. Recently, substantial advances have been made in the biodiscovery and engineering of superior nitroreductase variants, in particular development of elegant high-throughput screening capabilities to enable optimization of desirable activities via directed evolution. These advances in enzymology have been paralleled by advances in medicinal chemistry, leading to the development of second- and third-generation nitroaromatic prodrugs that offer substantial advantages over CB1954 for nitroreductase GDEPT, including greater dose-potency and enhanced ability of the activated metabolite(s) to exhibit a local bystander effect. In addition to forging substantial progress towards future clinical trials, this research is supporting other fields, most notably the development and improvement of targeted cellular ablation capabilities in small animal models, such as zebrafish, to enable cell-specific physiology or regeneration studies.

Advances in Enzymology and Related Areas of Molecular Biology Vol. 77. Edited by Eric J. Toone.

Advances in Enzymology and Related Areas of Molecular Biology Vol. 77. Edited by Eric J. Toone.

Specific Protein Patterns Characterize Metastatic Potential of Advanced Bladder Cancer

The prognosis in patients with metastasized bladder cancer is still poor. Clinical and histopathological parameters have limited ability to predict the risk of tumor progression. Thus, we identified specific protein patterns associated with tumor progression to differentiate specimens with and without metastasis. We analyzed 46 metastasized and 42 nonmetastasized muscle invasive bladder cancers by ProteinChip® technology surface enhanced laser desorption/ionization time of flight mass spectrometry. Cell lysis was done after laser capture microdissection from cryostat sections to achieve high tumor cell purity. Surface enhanced laser desorption/ionization time of flight mass spectrometry was completed with 2 matrices (Q10 and CM10). Bioinformatic analysis was performed by XLMiner® clustering using the Fuzzy c-means method. Differentially expressed proteins were identified and verified by 2-dimensional gel electrophoresis, tryptic in gel digest, peptide mapping, immunodepletion assay and Western blot analysis. By combining data on 2 chip surfaces (Q10 and CM10) results showed 86% sensitivity and 89% specificity in the training set, and 63% sensitivity and 88% specificity in the validation set. The relevant protein peaks 10.83, 14.68, 16.15 and 27.85 Da were identified as S100A8, MAP-1LC3, MUC-1S1 and GST-M1, respectively. We defined specific protein patterns with ProteinChip technology using bioinformatic evaluation software, which allowed differentiation between nonmetastasized and metastasized bladder tumor samples with high sensitivity and specificity. We identified 4 differentially expressed proteins. Thus, it seems possible to identify patients at high metastasized risk even at a clinically localized stage, leading to individual therapy decisions.

Application of enzymes for textile fibres processing

This review highlights the use of enzymes in the textile industry, covering both current commercial processes and research in this field. Amylases have been used for desizing since the middle of the last century. Enzymes used in detergent formulations have also been successfully used over the past 40 years. The application of cellulases for denim finishing and laccases for decolourization of textile effluents and textile bleaching are the most recent commercial advances. New developments rely on the modification of natural and synthetic fibres. Advances in enzymology, molecular biology and screening techniques provide possibilities for the development of new enzyme-based processes for a more environmentally friendly approach in the textile industry.

Lantibiotics: Peptides of Diverse Structure and Function

The current need for antibiotics with novel target molecules has coincided with advances in technical approaches for the structural and functional analysis of the lantibiotics, which are ribosomally synthesized peptides produced by gram-positive bacteria. These peptides have antibiotic or morphogenetic activity and are structurally defined by the presence of unusual amino acids introduced by posttranslational modification. Lantibiotics are complex polycyclic molecules formed by the dehydration of select Ser and Thr residues and the intramolecular addition of Cys thiols to the resulting unsaturated amino acids to form lanthionine and methyllanthionine bridges, respectively. Importantly, the structural and functional diversity of the lantibiotics is much broader than previously imagined. Here we discuss this growing collection of molecules and introduce some recently discovered peptides, review advances in enzymology and protein engineering, and discuss the regulatory networks that govern the synthesis of the lantibiotics by the producing organisms.

Protein-Derived Cofactors. Expanding the Scope of Post-Translational Modifications

Recent advances in enzymology, structural biology, and protein chemistry have extended the scope of the field of cofactor-dependent enzyme catalysis. It has been documented that catalytic and redox-active prosthetic groups may be derived from post-translational modification of amino acid residues of proteins. These protein-derived cofactors typically arise from the oxygenation of aromatic residues, covalent cross-linking of amino acid residues, or cyclization or cleavage of internal amino acid residues. In some cases, the post-translation modification is a self-processing event, whereas in others, another processing enzyme is required. The characterization of protein-derived cofactors and their mechanisms of biogenesis introduce a new dimension to our current views about protein evolution and protein structure-function relationships.

Acetylcholinesterase: Mechanism of Catalysis and Inhibition

Recent advances in the study of the catalytic properties of acetylcholinesterases have been reviewed. The main biological function of this enzyme is the fast termination of impulse transmission at cholinegric synapses by rapid hydrolysis of the neurotransmitter acetylcholine. Acetylcholinesterase has been often characterized as a perfect enzyme because its catalytic properties have been tuned to the highest possible limit. However, it seems paradoxical that the active site of this enzyme is buried deeply inside the enzyme molecule in the bottom of a narrow gorge restricting the traffic of substrates and products. The analysis of recent advances in enzymology and data on cholinesterase revealed rationality of this organization. The primary task of an enzyme catalyst is to lower the activation barrier for the chemical transformation of the substrate, and the catalytic power of the enzyme originates in polar solvation of the transition state by properly oriented dipoles inside the enzyme molecule. The a ctive site gorge of acetylcholinesterase, containing multiple potential substrate binding areas, is responsible for trapping and delivery of substrate molecules to the active site. The phenomena of allosteric modulation and substrate inhibition arise as secondary effects of the presence of the narrow gorge lined with hydrophobic residues. Keywords: Acetylcholinesterase, cholinesterases, SUBSTRATE HYDROLYSIS, ALLOSTERIC MODULATION, fasciculin-AChE

Thioflavin T Is a Fluorescent Probe of the Acetylcholinesterase Peripheral Site That Reveals Conformational Interactions between the Peripheral and Acylation Sites

Three-dimensional structures of acetylcholinesterase (AChE) reveal a narrow and deep active site gorge with two sites of ligand binding, an acylation site at the base of the gorge, and a peripheral site near the gorge entrance. Recent studies have shown that the peripheral site contributes to catalytic efficiency by transiently binding substrates on their way to the acylation site, but the question of whether the peripheral site makes other contributions to the catalytic process remains open. A possible role for ligand binding to the peripheral site that has long been considered is the initiation of a conformational change that is transmitted allosterically to the acylation site to alter catalysis. However, evidence for conformational interactions between these sites has been difficult to obtain. Here we report that thioflavin T, a fluorophore widely used to detect amyloid structure in proteins, binds selectively to the AChE peripheral site with an equilibrium dissociation constant of 1.0 microm. The fluorescence of the bound thioflavin T is increased more than 1000-fold over that of unbound thioflavin T, the greatest enhancement of fluorescence for the binding of a fluorophore to AChE yet observed. Furthermore, when the acylation site ligands edrophonium or m-(N, N,N-trimethylammonio)trifluoroacetophenone form ternary complexes with AChE and thioflavin T, the fluorescence is quenched by factors of 2.7-4.2. The observation of this partial quenching of thioflavin T fluorescence is a major advance in the study of AChE for two reasons. First, it allows thioflavin T to be used as a reporter for ligand reactions at the acylation site. Second, it indicates that ligand binding to the acylation site initiates a change in the local AChE conformation at the peripheral site that quenches the fluorescence of bound thioflavin T. The data provide strong evidence in support of a conformational interaction between the two AChE sites.

Evaluating the Safety of Microbial Enzyme Preparations Used in Food Processing: Update for a New Century

Microbial enzymes used in food processing are typically sold as enzyme preparations that contain not only a desired enzyme activity but also other metabolites of the production strain, as well as added materials such as preservatives and stabilizers. The added materials must be food grade and meet applicable regulatory standards. The purpose of this report is to present guidelines that can be used to evaluate the safety of the metabolites of the production strain that are also present in the enzyme preparation, including of course, but not limited to, the desired enzyme activity itself. This discussion builds on previously published decision tree mechanisms and includes consideration of new genetic modification technologies, for example, modifying the primary structure of enzymes to enhance specific properties that are commercially useful. The safety of the production strain remains the primary consideration in evaluating enzyme safety, in particular, the toxigenic potential of the production strain. Thoroughly characterized nonpathogenic, nontoxigenic microbial strains, particularly those with a history of safe use in food enzyme manufacture, are logical candidates for generating a safe strain lineage, through which improved strains may be derived via genetic modification by using either traditional/classical or rDNA strain improvement strategies. The elements needed to establish a safe strain lineage include thoroughly characterizing the host organism, determining the safety of all new DNA that has been introduced into the host organism, and ensuring that the procedure(s) that have been used to modify the host organism are appropriate for food use. Enzyme function may be changed by intentionally altering the amino acid sequence (e.g., protein engineering). It may be asked if such modifications might also affect the safety of an otherwise safe enzyme. We consider this question in light of what is known about the natural variation in enzyme structure and function and conclude tha t it is unlikely that changes which improve upon desired enzyme function will result in the creation of a toxic protein. It is prudent to assess such very small theoretical risks by conducting limited toxicological tests on engineered enzymes. The centerpiece of this report is a decision tree mechanism that updates previous enzyme safety evaluation mechanisms to accommodate advances in enzymology. We have concluded that separate mutagenicity testing is not needed if this decision tree is used to evaluate enzyme safety. Under the criteria of the decision tree, no new food enzyme can enter the market without critical evaluation of its safety.

PLASMA GLUTATHIONE S-TRANSFERASE π1-1 AND α1-1 LEVELS IN PATIENTS WITH BLADDER CANCER

No AccessJournal of UrologyINVESTIGATIVE UROLOGY1 Dec 2000PLASMA GLUTATHIONE S-TRANSFERASE π1-1 AND α1-1 LEVELS IN PATIENTS WITH BLADDER CANCER CHRIS L. BERENDSEN, THEO P.J. MULDER, and WILBERT H.M. PETERS CHRIS L. BERENDSENCHRIS L. BERENDSEN More articles by this author , THEO P.J. MULDERTHEO P.J. MULDER More articles by this author , and WILBERT H.M. PETERSWILBERT H.M. PETERS More articles by this author View All Author Informationhttps://doi.org/10.1016/S0022-5347(05)66982-0AboutFull TextPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract Purpose: Transitional cell carcinomas of the human bladder and many gastrointestinal tumors often contain high amounts of the detoxification enzyme glutathione S-transferase π (GSTP1-1). Elevated levels of GSTP1-1 have been found in serum and plasma from patients with gastrointestinal, lung or head and neck cancer. GSTP1-1 and glutathione S-transferase α (GSTA1-1) have been reported to be increased in 10 of 15 patients (67%) with bladder cancer. We evaluate the role of GSTP1-1 and GSTA1-1 as plasma tumor markers in 50 patients with bladder cancer before and after treatment. Materials and Methods: Blood from patients with bladder cancer was sampled in ethylenediaminetetraacetic acid tubes. Plasma GSTA1-1 and GSTP1-1 were measured using the sensitive and specific sandwich enzyme-linked immunosorbent assay. Results: Respective plasma GSTA1-1 and GSTP1-1 levels were above the upper normal reference limit in 2 (4%) and 14 (28%) of the 50 patients with bladder cancer. No significant decrease in plasma GSTA1-1 or GSTP1-1 was noted in matched pairs of plasma samples collected before and after treatment. Conclusions: In contrast to earlier reports, only a limited number of patients with bladder cancer had increased plasma GSTA1-1 or GSTP1-1, which did not decrease after tumor resection. These findings argue against the use of GSTP1-1 or GSTA1-1 as plasma markers for bladder cancer. References 1 : The glutathione S-transferase supergene family: regulation of GST and the contribution of the isoenzymes to cancer chemoprotection and drug resistance. Crit Rev Biochem Mol Biol1995; 30: 445. Google Scholar 2 : Nomenclature for human glutathione transferases. Biochem J1992; 282: 305. Google Scholar 3 : Preparation, characterization and properties of glutathione S-transferases. In: . Edited by . New York: Wiley J and Sons1985: 297. Google Scholar 4 : The isoenzymes of glutathione S-transferase. In: Advances in Enzymology. Edited by . New York: Wiley J and Sons1985: 357. Google Scholar 5 : Glutathione S-transferases: biomedical applications. Adv Clin Chem1993; 30: 282. Google Scholar 6 : Glutathione transferases and cancer. Crit Rev Biochem Mol Biol1992; 27: 337. 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Google Scholar From the Department of Urology, Atrium Medical Center Heerlen, Heerlen and Department of Gastroenterology, University Hospital St. Radboud, Nijmegen, The Netherlands© 2000 by American Urological Association, Inc.FiguresReferencesRelatedDetails Volume 164Issue 6December 2000Page: 2126-2128 Advertisement Copyright & Permissions© 2000 by American Urological Association, Inc.Keywordsglutathione transferasetumor markers, biologicalenzyme-linked immunosorbent assaybladder neoplasmsMetricsAuthor Information CHRIS L. BERENDSEN More articles by this author THEO P.J. MULDER More articles by this author WILBERT H.M. PETERS More articles by this author Expand All Advertisement PDF downloadLoading ...

Current approaches to the management of primary hyperoxaluria

Primary hyperoxalurias (PH) are very rare diseases characterised by overproduction and accumulation of oxalate in the body. The main target organ is the kidney, as oxalate cannot be metabolised and...

Turning Points in Catalysis

AbstractThis personal journey through the enlarging landscape of heterogeneous catalysis, pure and applied, starts with the discovery in 1823 of a dramatic example of the synthesis of water that was to serve as the basis of the first commercial exploitation of catalysis, Dobereiners lighter (tinder box). The quickening pace of successive industrial innovations and of academic insight during the intervening 170 years are summarized and the turning points, both major and minor, identified. Although new concepts and new ideas emerged in relative profusion, few exhibited the longevity predicted for them at birth by their proponents. Some concepts, like broken milestones on a vanished road, have ceased to retain their significance. Some have survived, even flourished. But in catalysis, as in most other branches of natural science, new tools and new techniques, rather than new concepts, tend to hold sway. And just as experimental advances in enzymology and immunology have led to the design of remarkable new biocatalysts so also, but not yet at the same level of delicate control, have the tools and techniques of solid‐state chemistry (including novel preparative and computational procedures), generated many powerful rationally designed inorganic catalysts.

Advances in enzymology and related areas of molecular biology (vol. 60)

Advances in enzymology and related areas of molecular biology (vol. 60)

Structure of a mycobacterial polysaccharide-fatty acyl-CoA complex: nuclear magnetic resonance studies.

MMP, a linear alpha 1 leads to 4 linked polymer of 3-O-methylmannose, regulates the fatty acid synthetase from Mycobacterium smegmatis by forming stoichiometric complexes with the long-chain acyl-CoA synthetase products. In agreement with previous proposals [Bloch, K. (1977) in Advances in Enzymology and Related Areas of Molecular Biology, ed. Meister, A. (Wiley, New York), Vol. 45, pp. 1-84], nuclear magnetic resonance studies show that the polysaccharide, a random coil in its free form, undergoes a major conformational transition upon enclosing long-chain acyl-CoA. The polysaccharide, probably in helical conformation in the complexed form, interacts with both the paraffinic chain and the CoA moieties of the included fatty acyl thioester.

Molecular mechanism of protein biosynthesis and an approach to the mechanism of energy transduction.

In 1941 Fritz Lipmann wrote an article in Advances in Enzymology which is now regarded as a landmark in the history of biochemistry. In this review he contemplated his new concept that the phosphate bonds serve as general carriers in energy transformation and in biosynthesis. The concept of “phosphate bond energy” or “energy-rich phosphate bond” has promptly prevailed among biochemists, and since then much effort has been focused on the mechanism of “group transfer” reactions in which the phosphate bond energy is very often utilized for the synthesis of covalent bonds.