The Important Role of Covalent Anchor Positions in Tuning Catalytic Properties of a Rationally Designed MnSalen-Containing Metalloenzyme (original) (raw)
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Metal-Conjugated Affinity Labels: A New Concept to Create Enantioselective Artificial Metalloenzymes
ChemistryOpen, 2013
Dedicated to Professor Wolfgang A. Herrmann on the occasion of his 65 th birthday Incorporation of artificial metal centers into proteins and peptides has emerged as an important tool in chemical and biological research. Current applications include pharmaceuticals, probes for molecular imaging [3] and contrast agents, [4] tools for biophysical studies targeting metalloprotein functions, metal-directed protein assembly, electrochemical biosensors, and altered electrochemical potential of electron transporting proteins, [8] as well as the synthesis of functional metalloenzymes with non-natural catalytic activity. [9] Particularly artificial metalloenzymes received great interest, since they hold the promise to greatly expand the range of reactions accessible by biocatalysis. A variety of methods to generate artificial metal sites were developed including domain-based directed evolution strategies, [10] engineering of transition-metal binding sites through introduction of coordinating amino acids at geometrically appropriate positions or site-directed in vivo incorporation of artificial metal-chelating amino acids. [12] However, the site-directed anchoring of artificial cofactors representing appropriate ligands or metal complexes has so far been the most successful strategy to achieve good catalytic activities and enantioselectivities. Inspired by the pioneering work of Wilson and Whitesides, Ward revealed the potential of the supramolecular biotin-(strept)avidin technology, which in combination with directed or rationally guided evolution can deliver highly enantioselective organometallic enzyme hybrid (OMEH) catalysts. [15] Such biotin-(strept)avidin-metal conjugates were subsequently tested in a variety of catalytic transformations. [16] In contrast to this supramolecular approach, covalent anchoring of artificial cofactors on proteins can utilize a variety of protein hosts and hence is not limited by the stability range of the biotin-(strept)avidin complex. Originally introduced by Kaiser, covalent attachment of artificial cofactors has been applied to convert proteases, [19] lipases [20] or other non-metal proteins [21] into organometallic enzyme hybrids. However, none of the covalent approaches could so far achieve the enantioselectivities reached by biotin-(strept)avidin conjugates. [9d, 21e] Although metals are introduced site-specifically, most systems generated through a covalent approach possess a flexible linker and hence lack a well-defined localization of the metal center on the surface of the host protein, which is a prerequisite to achieve chiral induction.
Design of artificial metalloenzymes
Applied Organometallic Chemistry, 2005
Homogeneous and enzymatic catalysis offer complementary means to generate enantiomerically pure compounds. For this reason, in a biomimetic spirit, efforts are currently under way in different groups to design artificial enzymes. Two complementary strategies are possible to incorporate active organometallic catalyst precursors into a protein environment. The first strategy utilizes covalent anchoring of the organometallic complexes into the protein environment. The second strategy relies on the use of non-covalent incorporation of the organometallic precursor into the protein. In this review, attention is focused on the use of semisynthetic enzymes to produce efficient enantioselective hybrid catalysts for a given reaction. This article also includes our recent research results and implications in developing the biotin-avidin technology to localize the biotinylated organometallic catalyst precursor within a well-defined protein environment.
Surprising cofactors in metalloenzymes
Current Opinion in Structural Biology, 2003
Transition metal complexes are located at the active sites of a number of enzymes involved in intriguing biochemical reactions. These complexes can exhibit a wide variety of chemical reactivity due to the ease at which transition metals can adopt different coordination environments and oxidation states. Crystallography has been a powerful technique for examining the structure and conformational variability of complex biological metallocenters. In particular, the past ten years have provided a wealth of structural information and several surprises concerning the metallocenters at the active sites of nitrogenase, hydrogenase and carbon monoxide dehydrogenase/acetyl-coenzyme A synthase.
Artificial metalloenzymes: proteins as hosts for enantioselective catalysis
Chemical Society Reviews, 2005
Enantioselective catalysis is one of the most efficient ways to synthesize high-added-value enantiomerically pure organic compounds. As the subtle details which govern enantioselection cannot be reliably predicted or computed, catalysis relies more and more on a combinatorial approach. Biocatalysis offers an attractive, and often complementary, alternative for the synthesis of enantiopure products. From a combinatorial perspective, the potential of directed evolution techniques in optimizing an enzyme's selectivity is unrivaled. In this review, attention is focused on the construction of artificial metalloenzymes for enantioselective catalytic applications. Such systems are shown to combine properties of both homogeneous and enzymatic kingdoms. This review also includes our recent research results and implications in the development of new semisynthetic metalloproteins for the enantioselective hydrogenation of N-protected dehydroamino acids.
Development of artificial metalloenzymes via covalent modification of proteins
2010
II.4.7 Chemical modification of proteins a. Conjugation with carboxylic acid functionalized phosphanes 1-4 b. Covalent modification with N-(4-(diphenylphosphane sulfide)benzyl) maleimide c. Coupling of maleimide functionalized azide and alkynes 5-7 d. Attempts to introduce BH3 protected phosphanes into alkyne and azide functionalized proteins via click-reaction e. Covalent modification with maleimide functionalized phosphaneboranes 10-12 f. Procedures tested for deprotection of phosphane-boranes coupled to proteins g. Coupling of phosphane ligands via hydrazone linkage III. Site-selective bioconjugation of nitrogen-containing ligands to structurally diverse protein hosts Firstly, I would like to thank my PhD supervisor, Prof. Paul Kamer for entrusting me with this interesting and challenging project, for his encouragement, help and academic supervision. I am also grateful to Prof. Ron Wever and Dr Paul Wright for agreeing to be the coexaminers in my dissertation committee. Special thanks to Dr Wouter Laan for his guidance during these years, for being a very demanding, rigorous and constructive scientific supervisor and for kindly proofreading the thesis. I would like to thank Prof Garry Taylor for allowing the use of laboratory space and equipment at the Centre for Biomolecular Sciences-St Andrews. I would like to acknowledge the BSRC Mass Spectrometry and Proteomics Department team, in particular Dr Catherine Botting and Dr Sally Shirran for helpful discussions and technical support in performing the LC-MS measurements and also Mr. Alex Houston for the MALDI TOF measurements. I would also like to thank the Solution-phase NMR Spectroscopy Service team, Dr Tomas Lebl and Mrs Melanja Smith for their help for the NMR measurements. I am thankful to Prof. Malcolm White for allowing us to perform the fluorescence measurements in his laboratory. I acknowledge the EPSRC National Mass Spectrometry Service Centre-Swansea for the accurate mass measurements performed there. I thank Prof. David A. Bernlohr (University of Minnesota) for kindly providing the pRSET-ALBP plasmid, as well as Dr Julio J. Caramelo (Fundación Instituto Leloir and Instituto de Investigaciones Bioquímicas de Buenos Aires), for kindly providing the pET22b(+) Δ98Δ IFAB plasmid. This work was financially supported by European Union Marie Curie Excellence Grants (MEXT-2004-014320). I am very thankful to all former and current colleagues from the Hom-Cat group. I have to start with thanks for our special colleague Bert, for his joyfulness and v brightness. Special thanks for Serghei, Bianca, Gregorio, Peter, Arnald, Debbie and Jason for their fruitful discussions and help with the organic chemistry project but also for being great friends and exceptional lab mates. I am indebted also to Yanmei and Nikos for the synthetic chemistry part. I want to thank Christine, Tanja and Michiel for the good and efficient time we spent together as a team. I am especially grateful to the Applecross girls for the breath of fresh air… provided every day when it was crucially needed. I feel very lucky and fulfilled for having the chance to meet interesting people and to make great friends here in the Chemistry Department, and I must thank Jenny, Bianca, Marzia, Aga, Jacorien and Louise for their permanent encouragements, support and friendship. Special thanks also for Elena, Alma, Cristian, Dragos and Andrei, for bringing into St Andrews the flavour of home. Many thanks also to Prof. Mircea Bulancea and Prof. Rodica Segal for their trust and encouragements at the beginning of my career. Without their initial support I would not be here today. Most importantly, I am grateful to my very special and loving family, for their permanent support. Special thanks to my sister for finding the strength to encourage and help me whilst she has to deal with her own life threatening problems, for my brother for being such a helpful, reliable and honest person and to my parents for teaching me through their personal examples the real values of life. Particular thanks to my daughter, who bravely started a new life in harsh conditions. I have to thank you for being so mature and determined in your work and for being such a good, patient and understanding person during my worst and best days here. What does not kill you makes you stronger and we are both alive! Last but not least, I want to thank God for giving me a second chance in life! vi
Artificial Metalloenzymes for Enantioselective Catalysis: Recent Advances
ChemBioChem, 2006
. Artificial metalloenzymes for enantioselective catalysis based on the incorporation of a catalytically active metal fragment within a host protein. The interaction between the metal fragment and the host protein may variously be supramolecular, dative, or covalent in nature.
From Unnatural Amino Acid Incorporation to Artificial Metalloenzymes
2016
Studies and development of artificial metalloenzymes have developed into vibrant areas of research. It is expected that artificial metalloenzymes will be able to combine the best of enzymatic and homogenous catalysis, that is, a broad catalytic scope, high selectivity and activity under mild, aqueous conditions. Artificial metalloenzyme consist of a host protein and a newly introduced artificial metal center. The host protein merely functions as ligand controlling selectivity and augmenting reactivity, while the metal center determines the reactivity. Potential applications range from catalytic production of fine chemicals and feedstock to electron transfer utilization (e.g. fuel cells, water splitting) and medical research (e.g. metabolic screening). Particularly modern asymmetric synthesis is expected to benefit from a successful combination of the power of biocatalysis (substrate conversion via multi-step or cascade reactions, potentially immortal catalyst, unparalleled selectivity and optimization by evolutionary methods) with the versatility and mechanism based optimization methods of homogeneous catalysis. However, so far systems are either limited in structural diversity (biotin-avidin technology) or fail to deliver the selectivities expected (covalent approaches). This thesis explores a novel strategy based on the site-selective incorporation of unnatural, metal binding amino acids into a host protein. The unnatural amino acids can either serve directly as metal binding centers can be used as anchoring points for artificial metallo-cofactors. The identification expression, purification and modification of a suitable protein scaffolds
Journal of The Royal Society Interface, 2014
In recent years, the design of artificial metalloenzymes obtained by the insertion of homogeneous catalysts into biological macromolecules has become a major field of research. These hybrids, and the corresponding X-ray structures of several of them, are offering opportunities to better understand the synergy between organometallic and biological subsystems. In this work, we investigate the resting state and activation process of a hybrid inspired by an oxidative haemoenzyme but presenting an unexpected reactivity and structural features. An extensive series of quantum mechanics/molecular mechanics calculations show that the resting state and the activation processes of the novel enzyme differ from naturally occurring haemoenzymes in terms of the electronic state of the metal, participation of the first coordination sphere of the metal and the dynamic process. This study presents novel insights into the sensitivity of the association between organometallic and biological partners and illustrates the molecular challenge that represents the design of efficient enzymes based on this strategy.