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Structural Studies on Dehydrogenases

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Structural Studies on Dehydrogenases Structural studies on dehydrogenases A thesis submitted in part fulfillment of the requirements for the degree of Doctor of Philosophy By Abdelhamid Elbrghathi B.Sc. (Hons) and M.Sc. University of Benghazi Department of Molecular Biology and Biotechnology University of Sheffield (July2014) Abstract The amino acid dehydrogenase superfamily has good potential for biotechnological use for example, either as enzymes for the production of non- natural amino acids, or for use as biosensors. Despite many years work on this family, there is still a gap understanding aspects of both substrate and cofactor binding and reaction mechanism. The crystal structures of M. smegmatis Glutamate dehydrogenase (GluDH) have been determined in two crystal forms, one of a ternary complex (GluDH/NADP+/2oxoglutarate) at 1.64Å, and one of a binary complex (GluDH/NADPH) at 1.78Å. The binary complex structures of C. symbiosum GluDH, with NAD+ and L-glutamate have also been solved at 1.80 Å and 1.24Å respectively. Comparison of these structures has allowed the nature of coenzyme specificity in GluDHs to be structurally characterized. Analysis of M. smegmatis GluDH/NADP+/2oxoglutarate ternary complex showed that 2- oxoglutarate bound to the enzyme as the gem-diol in an extended conformation, which mimics the structure of the carbinolamine intermediate. This structure, together with the high resolution C. symbiosum GluDH/L- glutamate complex structure, where the substrate binds in a compact, curved conformation have given valuable insights into the progress of the reaction. They show that the substrate alters its conformation during the reaction, explaining how the abortive reduction of the keto acid to the hydroxy acid is prevented, and showing the stereochemistry of attack by ammonia on the keto acid substrate, aspects of the mechanism that have been poorly understood. In addition, the structure of the AADH family member Bacillus sphaericus phenylalanine dehydrogenase in complex with NADH has been determined. Furthermore, initial experiments towards the structure of mycobacterium smegmatis typeII NADH: menaquinone oxidoreductase, which is a potential anti bacterial drug target are described. I Acknowledgement I would like to express my thanks and gratitude to my supervisor Dr. Patrick Baker for the great support, assistance, patience and encouragement during my PhD. I would also like to thank Prof David Hornby for all his help with explaining lots of molecular biology problems, and Dr Qaiser for his help with molecular biology lab. I would like to thank Dr Svetlana Sedelnikova for helping me in the protein purification experiments and crystallography group members who have assisted me during my period of study. All thanks going out to Libyan ministry of higher education for their financial support. I would like to thank my mum for her help and support all the time. I would also like to thank my wife for her support and encouragement. II List of Abbreviations Crystallographic Å Ångström a,b,c Real space unit cell dimension a* b* c* Reciprocal space unit cell dimension α,β,γ Real space unit cell angles B-factor Crystallographic temperature factor F(S) Structure factor Fcalc Calculated structure factors |Fc| Calculated structure factor amplitudes Fobs Observed structure factor F (hkl) Structure factor for reflection hkl |F (hkl)| Structure factor amplitude reflection hkl h, k, l Reciprocal lattice points I Diffraction intensity λ wavelength ρ(xyz) Electron density at position xyz Rfree Free R-factor Rmerge R-factor relating agreement between symmetry related reflections Rmsd Root mean square deviation Biological and chemical DEAE Diethylaminoethyl DNA Deoxyribonucleic acid dNTP Deoxyribonucleotide EDTA Ethylenediaminetetraacetic acid IPTG Isopropyl β- D-1-thiogalactopyranoside MES 2-(N-morpholino)ethanesulfonic acid mRNA Messenger ribonucleic acid NADPH Nicotinamide adenine dinucleotide phosphate PEG Polyethylene glycol RNase RNA nuclease III SDS Sodium dodecyl sulfate NADH Nicotinamide adenine dinucleotide NADPH Nicotinamide adenine dinucleotide phosphate GluDH Glutamate dehydrogenase PheDH Phenylalanine dehydrogenase LeuDH Leucine dehydrogenase List of organisms Mycobacterium smegmatis M. smegmatis Clostridium symbiosum C. symbiosum Bacillus sphaericus B. sphaericus Escherichia coli E. coli IV Table of content Chapter one: Introduction ..................................................................................................... 1 1.1 Amino acid dehydrogenases ................................................................................................... 1 1.1.1 Glutamate dehydrogenase (GluDH) .............................................................................................. 2 1.1.2 Phenylalanine dehydrogenase ......................................................................................................... 2 1.2 The biological function of amino acid dehydrogenases ................................................ 3 1.2.1 Biological role of GluDHs .................................................................................................................... 3 1.2.1.1 Ammonia assimilation ................................................................................................................................ 4 1.2.1.2 Amino acid degradation ............................................................................................................................. 6 1.3 Molecular weight and quaternary structure ..................................................................... 6 1.4 Structures and conformational changes of amino acid dehydrogenases ................ 8 1.4.1 Structures of glutamate dehydrogenase ..................................................................................... 8 1.4.1.1 Bacterial GluDHs ............................................................................................................................................ 8 1.4.1.2 Mammalian GluDHs ...................................................................................................................................... 9 1.4.1.3 Conformational change of GDHs ............................................................................................................. 9 1.4.2 Structure of Leucine dehydrogenase ......................................................................................... 10 1.4.3 PheDH structure ................................................................................................................................. 13 1.5 Coenzyme specificity of amino acid dehydrogenases .................................................. 15 1.5.1 Coenzyme stereospecificity ........................................................................................................... 17 1.6 Substrate specificity of amino acid dehydrogenases ................................................... 17 1.6.1 Glutamate dehydrogenase .............................................................................................................. 17 1.6.2 Phenylalanine dehydrogenase ...................................................................................................... 20 1.6.3 Leucine dehydrogenase ................................................................................................................... 20 1.7 Homology and sequence alignment of amino acid dehydrogenases ...................... 21 1.8 Chemical mechanism of GluDHs .......................................................................................... 22 1.9 Chemical mechanism for phenylalanine dehydrogenase ........................................... 27 1.10 Previous structures of amino acid dehydrogenases .................................................. 29 1.11 Project Aim ............................................................................................................................... 29 Chapter Two: Materials and Methods ............................................................................. 36 2.0 Overview ............................................................................................................................ 36 2.1 Gene amplification, cloning and overexpression of glutamate dehydrogenase from M. smegmatis .......................................................................................................................... 36 2.1.1 The design of primers ....................................................................................................................... 36 2.1.2 Amplification of target gene using polymerase chain reaction (PCR) ......................... 36 2.1.2.1 Purification of PCR products from agarose gel .............................................................................. 38 V 2.1.3 Preparation of pET21a plasmid ................................................................................................... 38 2.1.4 Restriction digestion ......................................................................................................................... 39 2.1.5 Ligation of PCR product into plasmid ........................................................................................ 40 2.1.5.1 Transformation of ligation mixes into DH5α E. coli ..................................................................... 40 2.1.5.2 Screening of possible clones ................................................................................................................... 41 2.1.6 Protein Expression ............................................................................................................................
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