The articles of the conference proceedings are published in Perspectives in Science (Volume 9, Pages 1-70, December 2016) and freely accessible.
Proceedings of the
7th Beilstein ESCEC Symposium
From Enzymology to Systems Biology and Back
14 – 18 September 2015, Rüdesheim, Germany
From enzymology to systems biology and back – Prolog
Carsten Kettner and Martin G. Hicks
Beilstein-Institut, Frankfurt am Main, Germany
About twenty years ago, systems biology was far away from modeling large metabolic networks due to insufficient computational power to go beyond modeling the reaction kinetics of individual enzymes. Today, after the enormous increases in the speed of computation and growth in data storage capabilities, systems biology is approaching its goal to be able to investigate complex biological systems be it individual cells, tissues, organs or even whole organisms. Furthermore, in systems biology, modeling of pathways not only includes single enzyme reaction kinetics but also higher-level controlling processes such as gene regulation and signal transduction making the in-silico reconstruction of such heterogeneous networks significantly more complicated...
Finding homes for orphan enzymes
Frank M. Raushel
Department of Chemistry, Texas A&M University, College Station, TX, USA
The rate at which new genes are being sequenced greatly exceeds our ability to correctly annotate the functional properties of the corresponding proteins. Annotations based primarily on sequence identity to experimentally characterized proteins are often misleading because closely related sequences may have different functions, while highly divergent sequences may have identical functions. Our understanding of the principles that dictate the catalytic properties of enzymes, based on protein sequence alone, is often insufficient to correctly annotate proteins of unknown function. To address these problems, we are working to develop a comprehensive strategy for the functional annotation of newly sequenced genes using a combination of structural biology, bioinformatics, computational biology, and molecular enzymology. The power of this multidisciplinary approach for discovering new reactions catalyzed by uncharacterized enzymes has been tested using the amidohydrolase superfamily as a model system.
In search of lost time constants and of non-Michaelis–Menten parameters
Maria F. Pinto1,2 and Pedro M. Martins1,2
1 ICBAS, Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto, Porto, Portugal
2 LEPABE, Laboratório de Engenharia de Processos, Ambiente, Biotecnologia e Energia, Departamento de Engenharia Química, Faculdade de Engenharia da Universidade do Porto, Porto, Portugal
Upon completing 100 years since it was published, the work Die Kinetik der Invertinwirkung by Michaelis and Menten (MM) was celebrated during the 6th Beilstein ESCEC Symposium 2013. As the 7th Beilstein ESCEC Symposium 2015 debates enzymology in the context of complex biological systems, a post-MM approach is required to address cell-like conditions that are well beyond the steady-state limitations. The present contribution specifically addresses two hitherto ambiguous constants whose interest was, however, intuited in the original MM paper: (i) the characteristic time constant τ∞, which can be determined using the late stages of any progress curve independently of the substrate concentration adopted; and (ii) the dissociation constant KS, which is indicative of the enzyme–substrate affinity and completes the kinetic portrayal of the Briggs–Haldane reaction scheme. The rationale behind τ∞ and KS prompted us to revise widespread concepts of enzyme's efficiency, defined by the specificity constant kcat/KM, and of the Michaelis constant KM seen as the substrate concentration yielding half-maximal rates. The alternative definitions here presented should help recovering the wealth of published kcat/KM and KM data from the criticism that they are subjected. Finally, a practical method is envisaged for objectively determining enzyme's activity, efficiency and affinity – (EA)2 – from single progress curves. The (EA)2 assay can be conveniently applied even when the concentrations of substrate and enzyme are not accurately known.
Reconstruction of ancestral enzymes
Rainer Merkl and Reinhard Sterner
University of Regensburg, Institute of Biophysics and Physical Biochemistry, Regensburg, Germany
The amino acid sequences of primordial enzymes from extinct organisms can be determined by an in silico approach termed ancestral sequence reconstruction (ASR). In the first step of an ASR, a multiple sequence alignment (MSA) comprising extant homologous enzymes is being composed. On the basis of this MSA and a stochastic model of sequence evolution, a phylogenetic tree is calculated by means of a maximum likelihood approach. Finally, the sequences of the ancestral proteins at all internal nodes including the root of the tree are deduced. We present several examples of ASR and the subsequent experimental characterization of enzymes as old as four billion years. The results show that most ancestral enzymes were highly thermostable and catalytically active. Moreover, they adopted three-dimensional structures similar to those of extant enzymes. These findings suggest that sophisticated enzymes were invented at a very early stage of biological evolution.
Tools and strategies for discovering novel enzymes and metabolic pathways
John A. Gerlt
Institute for Genomic Biology and Departments of Biochemistry and Chemistry, University of Illinois at Urbana–Champaign, Urbana, IL, USA
The number of entries in the sequence databases continues to increase exponentially – the UniProt database is increasing with a doubling time of ∼4 years (2% increase/month). Approximately 50% of the entries have uncertain, unknown, or incorrect function annotations because these are made by automated methods based on sequence homology. If the potential in complete genome sequences is to be realized, strategies and tools must be developed to facilitate experimental assignment of functions to uncharacterized proteins discovered in genome projects. The Enzyme Function Initiative (EFI; previously supported by U54GM093342 from the National Institutes of Health, now supported by P01GM118303) developed web tools for visualizing and analyzing (1) sequence and function space in protein families (EFI-EST) and (2) genome neighbourhoods in microbial and fungal genomes (EFI-GNT) to assist the design of experimental strategies for discovering the in vitro activities and in vivo metabolic functions of uncharacterized enzymes. The EFI developed an experimental platform for large-scale production of the solute binding proteins (SBPs) for ABC, TRAP, and TCT transport systems and their screening with a physical ligand library to identify the identities of the ligands for these transport systems. Because the genes that encode transport systems are often co-located with the genes that encode the catabolic pathways for the transported solutes, the identity of the SBP ligand together with the EFI-EST and EFI-GNT web tools can be used to discover new enzyme functions and new metabolic pathways. This approach is demonstrated with the characterization of a novel pathway for ethanolamine catabolism.
The bacterial catabolism of polycyclic aromatic hydrocarbons: Characterization of three hydratase-aldolase-catalyzed reactions
Jake A. LeVieux, William H. Johnson Jr., Kaci Erwin, Wenzong Li, Yan Jessie Zhang, Christian P. Whitman
Division of Chemical Biology and Medicinal Chemistry, College of Pharmacy, and the Department of Molecular Biosciences, The University of Texas, Austin, TX, USA
Polycyclic aromatic hydrocarbons (PAHs) are highly toxic, pervasive environmental pollutants with mutagenic, teratogenic, and carcinogenic properties. There is interest in exploiting the nutritional capabilities of microbes to remove PAHs from various environments including those impacted by improper disposal or spills. Although there is a considerable body of literature on PAH degradation, the substrates and products for many of the enzymes have never been identified and many proposed activities have never been confirmed. This is particularly true for high molecular weight PAHs (e.g., phenanthrene, fluoranthene, and pyrene). As a result, pathways for the degradation of these compounds are proposed to follow one elucidated for naphthalene with limited experimental verification. In this pathway, ring fission produces a species that can undergo a non-enzymatic cyclization reaction. An isomerase opens the ring and catalyzes a cis to trans double bond isomerization. The resulting product is the substrate for a hydratase-aldolase, which catalyzes the addition of water to the double bond of an α,β-unsaturated ketone, followed by a retro-aldol cleavage. Initial kinetic and mechanistic studies of the hydratase-aldolase in the naphthalene pathway (designated NahE) and two hydratase-aldolases in the phenanthrene pathway (PhdG and PhdJ) have been completed. Crystallographic work on two of the enzymes (NahE and PhdJ) provides a rudimentary picture of the mechanism and a platform for future work to identify the structural basis for catalysis and the individual specificities of these hydratase-aldolases.
Biocatalytic retrosynthesis: Redesigning synthetic routes to high-value chemicals
Anthony P. Green, Nicholas J. Turner
School of Chemistry, Manchester Institute of Biotechnology, University of Manchester, Manchester, UK
Modern tools for enzyme discovery combined with the development of increasingly reliable strategies for protein engineering have greatly expanded the range of enzymes with suitable properties for practical applications. This situation presents enormous opportunities for the design of sustainable biocatalytic strategies for the production of high-value chemicals. Here, we highlight recent contributions from our laboratory concerning ω-transaminases and monoamine oxidases, two enzyme classes that have been exploited for the industrial scale production of active pharmaceutical ingredients or key chiral intermediates. Firstly, we describe the development of novel ‘smart’ amine donors which overcome inherent challenges associated with controlling the equilibrium position of ω-TA catalyzed processes. Subsequently, we demonstrate how engineered variants of monoamine oxidase developed in our laboratory have been applied as biocatalysts for the synthesis of a diverse range of active pharmaceutical ingredients and alkaloid natural products. Through these illustrative examples, we hope to promote the wider application of enzymes within the synthetic community.
Computational approaches for the study of the role of small molecules in diseases
Institute of Bioinformatics and Systems Biology, Helmholtz Zentrum München, Neuherberg, Germany
An enormous amount of molecular and phenotypic information of drugs as well as diseases is now available in public repositories. Computational analysis of these datasets is facilitating the acquisition of a systems view of how drugs act on our human organism and interfere with diseases. Here, I highlight recent approaches integrating large-scale information of drugs and diseases that are contributing to change our current view on how drugs interfere with human diseases.
Identifying the conditions necessary for the thioredoxin ultrasensitive response
Johann M. Rohwer1, Charl Viljoen1, Carl D. Christensen1, Lefentse N. Mashamaite2, Ché S. Pillay2
1Laboratory for Molecular Systems Biology, Department of Biochemistry, Stellenbosch University, Stellenbosch, South Africa
2School of Life Sciences, University of KwaZulu-Natal, Carbis Road, Pietermaritzburg, South Africa
Thioredoxin, glutaredoxin, and peroxiredoxin systems (collectively called redoxins) play critical roles in a large number of redox-sensitive cellular processes. These systems are linked to each other by coupled redox cycles and by common reaction intermediates into a larger network.
Previous results from a realistic computational model of the Escherichia coli thioredoxin system developed in our group have revealed several modes of kinetic regulation in the system. Amongst others, the coupling of the thioredoxin and peroxiredoxin redox cycles was shown to exhibit the potential for ultrasensitive changes in the thioredoxin concentration and the flux through other thioredoxin-dependent processes in response to changes in the thioredoxin reductase level. Here, we analyse the basis for this ultrasensitive response using kinetic modelling and metabolic control analysis and derive quantitative conditions that must be fulfilled for ultrasensitivity to occur.
The enigmatic conservation of enzyme dynamics in evolution
Department of Chemistry, University of Iowa, Iowa City, IA, USA
Examination of the chemical step catalysed by dihydrofolate reductase (DHFR) suggested preservation of an “ideal” transition state as the enzyme evolves from bacteria to human. This observation is enigmatic: since evolutionary pressure is most effective on enzymes’ second order rate constant (kcat/KM) and since the chemistry is not rate limiting on that kinetic parameter, why is the nature of the chemical step preserved? Studies addressing this question were presented in the 2015 Beilstein ESCEC Symposium and are summarized below.
From enzymology to systems biology and back – Epilog
University of Strathclyde, Glasgow, Scotland, UK
In this report, I have tried to identify some common themes of the meeting, which span multiple talks. I also pick out a few highlights, as identified by other participants, using anonymous forms I circulated at the meeting. Some presenters’ names are mentioned, but this is purely for illustration. I do not wish to imply that other talks were not equally as informative and interesting. I hope this format will be more useful to readers than the commonly found pattern of a few sentences summarizing each talk – the presenters themselves offer such abstracts in the program and proceedings.