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Proceedings of the
5th Beilstein ESCEC Symposium
From Protein Structure to Enzyme Kinetics
The multi-disciplinary approaches which are now typical of research in the natural and life sciences use a combination of modern experimental techniques which have led to an increased accuracy in the measurements of enzyme structures and activities. Modern analysis methods often result in the generation of huge amounts of data and large data sets, which are subsequently published in electronic data repositories and in publications.
However, data in both the literature and in databases suffer from the fact that they are often noncomparable due to incomplete and imprecise descriptions of materials and methods. Furthermore, if the experimental conditions are not fully and accurately stated, the values of the functional data of enzyme activities will be of little use for applications such as systems biology. Further problems occur even when the data are well reported; they will have often been collected under quite disparate conditions so that researchers are faced with the problem of the range of method-specific enzyme data. This is often an issue when data move between researchers whose data are supplied by laboratories that use different methods, and can, in the worst case, lead to misinterpretation of laboratory findings.
Since 2003 the STRENDA Commission (Standards for Reporting Enzyme Data) has been actively working on concepts to improve the quality of reporting functional enzyme data that will allow the efficient use of enzyme kinetics in the in vivo, in vitro and in silico investigation of biological systems. The Commission has two major goals: the first is the development of a set of guidelines for the reporting of data in publications. These guidelines are currently recommended by 28 biochemistry journals. The second goal is the development of an electronic data submission tool that incorporates the STRENDA Guidelines, and which is intended to act as a portal for the submission of enzyme kinetics data to a freely-accessible, public database.
The previous four ESCEC symposia have not only supported the work of the STRENDA Commission, but have also lead to the symposium becoming established as a scientific meeting in its own right. For the fifth symposium, the organizers decided to choose 'Protein Structure Meets Enzyme Kinetics' as the focus of the meeting – providing a perfect example of an interesting and important area of contempory science where the reporting of data needs to be improved. The characterization of enzyme functions is usually accompanied by the determination of the rates by which enzymes catalyze reactions. This knowledge can give insights into the mechanism of the reaction, which in turn relates to the structure of the enzyme.
At the 5th ESCEC Symposium, organized by the Beilstein-Institut together with the STRENDA Commission, we were fortunate that a diverse range of speakers not only accepted our invitation to present but that most were able attend for the whole meeting and so participate in the lively discussions. Topics covered ranged from describing how the modification of enzyme structures affects the kinetics of enzyme reactions to discussing new results, approaches and methodologies for establishing physiological ties between sequence, structure and kinetics and modeled networks of collaborative enzymes. The overall topic – standard representation of enzyme data – was considered and discussed in precise detail when the initial version of the STRENDA capturing tool was presented. This has enabled the Commission to complete a working data acquisition prototype, which can be accessed at http://www.strenda.org/eform.html/. Any comments and suggestions are still welcome!
We would like to thank particularly the authors who provided us with written versions of the papers that they presented. Special thanks go to all those involved with the preparation and organization of the symposium, to the chairmen who piloted us successfully through the sessions, and to the speakers and participants for their contribution in making this symposium a valuable and fruitful event.
Frankfurt/Main, February 2013
Martin G. Hicks
Phosphonates to Phosphate: A Functional Annotation of the Essential Genes of the Phn Operon in Escherichia coli
Siddhesh S. Kamat, Howard J. Williams, and Frank M. Raushel
Department of Chemistry, Texas A&M University, College Station.
The reaction mechanism for the enzymatic conversion of methyl phosphonate to phosphate and methane in Escherichia coli has eluded researchers over the last three decades despite significant genetic and in vivo studies. The phn operon governs the C-P lyase activity in E. coli. The essential genes within the phn operon are phnGHIJKLM.
The proteins encoded by phnGHM were over-expressed in E. coli and purified to homogeneity using standard protocols. The proteins encoded by phnIJKL were soluble only when expressed as N-terminal glutathione S-transferase (GST) fusion proteins. PhnI was shown to catalyse the formation of α-D-ribose-1-methylphosphonate-5-triphosphate (RPnTP) from MgATP and methylphosphonate in the presence of PhnG, PhnH, and PhnL after in situ cleavage of the GST-tags.
PhnI alone catalyses the hydrolytic cleavage of MgATP to adenine and D-ribose-5-triphosphate (RTP). PhnM catalyses the hydrolysis of α-D-ribose-1-methylphosphonate-5-triphosphate (RPnTP) to α-D-ribose-1-methylphosphonate-5-phosphate (PRPn) and pyrophosphate with attack of water on the α-phosphoryl group of the triphosphate moiety of RPnTP. PhnJ was reconstituted with an iron-sulphur cluster through the anaerobic addition of FeSO4, Na2S and Na2S2O4 under strictly anaerobic conditions. The [Fe4S4]-reconstituted PhnJ GST-fusion protein catalyses the homolytic cleavage of the phosphorus-carbon bond of α-D-ribose-1-methylphosphonate-5-phosphate (PRPn) to ultimately form α-D-ribose-1,2-cyclic-phosphate-5-phosphate (PRcP) and methane in the presence of S-adenosyl-L-methionine (SAM) under strictly anaerobic conditions, after in situ cleavage of the GST-tag.
IMP Dehydrogenase: the Dynamics of Reaction Specificity
Departments of Biology and Chemistry, Brandeis University, Waltham.
Subtle changes in enzyme structure can have enormous impact on catalysis, as vividly illustrated in IMP dehydrogenase (IMPDH) and GMP reductase (GMPR). These proteins share a common structure and set of catalytic residues and bind the same ligands with similar affinities. IMPDH catalyses a hydride transfer reaction involving a nicotinamide cofactor, with formation of the covalent intermediate E-XMP*. Hydrolysis of this intermediate produces XMP, which is converted to GMP by the action of another enzyme. In the GMPR reaction, EXMP* is formed by the deamination of GMP, and is subsequently reduced via a hydride transfer reaction with a nicotinamide cofactor. In both cases, a conformational change separates the two chemical transformations. The protein moves in the case of IMPDH, while the cofactor moves in GMPR. Thus conformational dynamics control reaction specificity in the IMPDH/GMPR family, with intriguing implications for the evolution of these enzymes.
The pH-induced Metabolic Shift from Acidogenesis to Solventogenesis in Clostridium acetobutylicum – From Experiments to Models
Thomas Millat1, Holger Janssen2, Hubert Bahl2, Ralf-Joerg Fischer2 and Olaf Wolkenhauer1,3
1Department of Systems Biology & Bioinformatics, University of Rostock.
2Division of Microbiology, University of Rostock.
3Institute for Advanced Study (STIAS), Wallenberg Research Center, Stellenbosch
The strictly anaerobic Gram-positive Clostridium acetobutylicum is able to ferment starchy material to acetone, butanol, and ethanol. Due to rising costs, dwindling resources, and environmental concerns regarding extraction and use of petroleum and natural gas, the academic and industrial interest in C. acetobutylicum has been renewed in recent years. However, an improved understanding of the clostridial metabolism and its regulations is a prerequisite for future industrial applications.
The COSMIC consortium, as part of the transnational SysMo initiative, is focusing on the pH-induced metabolic shift of C. acetobutylicum from acidogenesis to solventogenesis. During acidogenesis (high pH) the bacterium predominantly produces the acids acetate and butyrate, whereas the solvents acetone and butanol are fermented during solventogenesis (low pH). This metabolic phase transition is accompanied by changes in transcriptome, proteome, and metabolome which have been measured using a standardized experimental setup.
The information gathered is used to model this dynamic shift. Towards this end, we established a system of coupled differential equations, describing the biochemical reactions involved in AB fermentation and their dynamic changes found in recent experiments. Since C. acetobutylicum is not capable of maintaining a homoeostatic intracellular pH, the influence of a changing intracellular pH on enzyme activity and stability is of special interest for an improved understanding of AB fermentation. Such a model is able to predict product spectrum and metabolome during the pH-induced shift as well as for several mutants at solventogenesis.
Slow-onset Enzyme Inhibition and Inactivation
Department of Biochemistry, University of Zurich.
Interactions between modifiers and enzymes can either occur rapidly, on the time scale of diffusion-controlled reactions, or they can be slow processes observable on the steady-state time scale. Slow interactions in hysteretic enzymes serve to dampen cellular responses to rapid changes in metabolite concentration as part of regulatory mechanisms. Naturally occurring inhibitors of several enzymes, such as the macromolecular proteinaceous inhibitors of peptidases, may act slowly when forming complexes with their targets. To allow physiologically meaningful rates of enzyme inhibition, the modifier concentration is kept at high levels in nature but problems arise when these levels drop for some reason. The slow-onset inhibitory behavior of enzyme modifiers used as drugs may represent a handicap if their concentration at the target site is insufficient and/or the kinetic constants are inadequate to warrant pharmacologically meaningful rates of enzyme inhibition. A truthful knowledge of mechanisms and kinetic constants of such systems is mandatory for making predictions on the efficiency of the modifiers in vivo.
Ontology-based search in SABIO-RK
Ulrike Wittig, Enkhjargal Algaa, Andreas Weidemann, Renate Kania, Maja Rey, Martin Golebiewski, Lei Shi, Lenneke Jong and Wolfgang Mueller
Scientific Databases and Visualization Group, Heidelberg Institute for Theoretical Studies (HITS), Heidelberg.
The SABIO-RK database (http://sabio.h-its.org/) is established as a resource for biochemical reactions and their kinetic data. Data are manually extracted from scientific literature and stored in a structured and standardised format. Additionally SABIO-RK allows direct submission of data from lab experiments in an automated workflow, e. g. within project collaborations for storage and exchange of unpublished experimental results and later publishing the data. To access the kinetic data in the database, web interfaces and web services are available offering complex searches using various criteria. For specific search criteria different classification levels of organisms, tissues, and reactants, can be selected based on biological ontologies. Biological ontologies are developed for a hierarchical classification of biological objects and for modelling a domain using shared vocabularies. Ontological relations are implemented in SABIO-RK to extend the search functionalities.
Protein Engineering Elucidates the Relationship between Structure, Function and Stability of a Metabolic Enzyme
Reinhard Sterner, Thomas Schwab and Sandra Schlee
Institute of Biophysics and Physical Biochemistry, University of Regensburg.
The relationship between oligomerisation state, stability, and catalytic activity of the anthranilate phosphoribosyl transferase from Sulfolobus solfataricus (sAnPRT) was analysed by three interrelated protein engineering approaches. The extremely thermostable homodimeric sAnPRT enzyme was converted into a monomer by rational design, and its low catalytic activity at 37 °C was elevated by a combination of random mutagenesis and metabolic selection in the mesophilic host Escherichia coli. The two amino acid exchanges leading to monomerization and the two substitutions resulting in activation of sAnPRT were then combined, which resulted in an "activated monomer" that was significantly less stable and more active than wild-type sAnPRT. Using a combination of random mutagenesis and selection in the thermophilic host Thermus thermophilus, the activated monomer was stabilized, and the consequences of stabilization for catalytic activity and association state were analysed.
Successes and Challenges in Functional Assignment in a Superfamily of Phosphatases
Karen N. Allen1 and Debra Dunaway-Mariano2
1Department of Chemistry, Boston University.
2Department of Chemistry and Chemical Biology, University of New Mexico, Albuquerque.
The explosion of protein sequence information from genome sequencing efforts requires that current experimental strategies for function assignment must evolve into computationally-based function prediction.
This necessitates the development of new strategies based, in part, on the identification of sequence markers, including residues that support structure and specificity as well as a more informed definition of orthologues. We have undertaken the function assignment of unknown members of the haloalkanoate dehalogenase superfamily using an integrated bioinformatics/structure/mechanism approach. Notably, a number of members show ‘‘substrate blurring’’, with activity toward a number of substrates and significant substrate overlap between paralogues. Other family members have been honed to a specific substrate with high catalytic efficiency and proficiency. Our findings highlight the use of the cap domain structure and enzyme conformational dynamics in delineating specificity.
Functional (Mis)Assignment in the Tomaymycin Biosynthetic Pathway
Christian P. Whitman
Division of Medicinal Chemistry, College of Pharmacy, The University of Texas at Austin
4-Oxalocrotonate tautomerase (4-OT) catalyses the conversion of 2-hydroxymuconate to 2-oxo-3-hexenedioate in microbial pathways for the degradation of aromatic hydrocarbons. Pro-1 functions as a general base and shuttles the 2-hydroxy proton to C-5 of the product. Two arginine residues, Arg-11 and Arg-39, facilitate the reaction by participating in binding and catalysis. The same reaction is carried out by a heterohexamer 4-OT (hh4-OT) in thermophilic bacteria. The α-subunit of the hh4-OT identified the 4-OT homologue TomN in the biosynthetic cluster for the C ring of the antitumor antibiotic tomaymycin. TomN shares 58% pairwise sequence similarity with 4-OT including the three key catalytic residues. Kinetic and mutagenesis studies show that TomN catalyses the canonical 4-OT reaction with comparable efficiency using the same mechanism. However, the proposed function for TomN involves a very different reaction from that carried out by 4-OT. These results suggest that the assignment for TomN and the sequence of events leading to the C ring of tomaymycin might not be correct.
Thermodynamic Characterisation of Carbohydrate-active Enzymes
Oliver Ebenhoeh1, Alexander Skupin2, Oender Kartal3, Sebastian Mahlow4 and Martin Steup4
1Institute for Complex Systems and Mathematical Biology, University of Aberdeen.
2Luxembourg Centre for Systems Biomedicine, University of Luxembourg, Luxembourg.
3Department of Biology, Group of Plant Biotechnology, ETH Zurich.
4Department of Plant Physiology, Institute of Biochemistry and Biology, University of Potsdam.
Many carbohydrate-active enzymes catalyse a specific reaction pattern instead of one particular reaction. For example, glucanotransferases and glucosyltransferases recognise the reducing ends of glucans irrespective of their degree of polymerisation. Thus, in principle, they are capable of catalysing an infinite number of reactions. Here we show how concepts from statistical thermodynamics can be employed to characterise the action patterns of polymer-active enzymes and determine their equilibrium distributions. For selected enzymes, we provide experimental evidence that our theory provides accurate predictions. We further outline how the thermodynamic description can be employed to experimentally determine bond energies from equilibrium distributions of polymer-active enzymes.
Metal Binding Sites in Proteins
Vladimir Sobolev, Ronen Levy, Mariana Babor and Marvin Edelman
Department of Plant Sciences, Weizmann Institute of Science, Rehovot.
Metal ions play a critical role in living systems. About one third of proteins need to bind metal for their stability and/or function. In this review, current sequence based and structure based methods for metal binding site prediction will be presented, with emphasis on the CHED and SeqCHED methods of prediction from apo-protein structures and protein sequences having homologs (even remote) in the structural protein databank (PDB). Metal binding site prediction will be considered as a step in function assignment for new proteins. Finally, a disproportional association of first and second shell metal binding residues in human proteins with disease-related SNPs will be shown.