Active Site and Loop Region CUREs
Multiple Roles of the Active Site and Loop Region of Malate Dehydrogenase:
Research project this CURE supports: ACTIVE SITE AND LOOP REGION
Description of the Science/background for this CURE:
The research theme at the heart of this CURE is a perennial debate over the roles that protein dynamics can play in enzyme function. Do protein dynamics play a direct role in hydride transfer reactions as has been proposed or in the electrostatic pre-organization of the active site . What role do protein dynamics play in conformational selection processes that may be involved in promiscuous substrate utilization. Many enzymes have Histidine-Aspartate pairs involved in catalysis and Malate Dehydrogenases universally contain a His-Asp diad involved in proton abstraction/donation from/to malate/oxaloacetate. The role of loop residues and active site second sphere residues in catalysis and substrate specificity is understudied.. Evolutionary relationships between Malate Dehydrogenases and Lactate Dehydrogenases. Adaptation of enzymes to extreme conditions includes roles of dynamics and second sphere residues in structure function relationships.
Relevant Literature that support this science:
General Background to Malate Dehydrogenase
Literature sources that support this science:
Dalziel K. Dynamic aspects of enzyme specificity. Philos Trans R Soc Lond B Biol Sci. 1975 Nov 6;272(915):109-22. doi: 10.1098/rstb.1975.0074. PMID: 1807.
Alexandra Roessling, Ellis Bell, Jessica Bell. The Role of the Flexible Loop in Substrate Recognition and Catalysis in Malate Dehydrogenase.: 18 April 2020., https://doi.org/10.1096/fasebj.2020.34.s1.05403
De Luca V, Mandrich L. Enzyme Promiscuous Activity: How to Define it and its Evolutionary Aspects. Protein Pept Lett. 2020;27(5):400-410. doi: 10.2174/0929866527666191223141205. PMID: 31868141.
Weikl, T.R. and Paul, F. (2014), Conformational selection in protein binding and function. Protein Science, 23: 1508-1518. https://doi.org/10.1002/pro.2539
Ma B, Nussinov R. Enzyme dynamics point to stepwise conformational selection in catalysis. Curr Opin Chem Biol. 2010 Oct;14(5):652-9. doi: 10.1016/j.cbpa.2010.08.012. Epub 2010 Sep 6. PMID: 20822947; PMCID: PMC6407632.
Campbell, E., Kaltenbach, M., Correy, G. et al. The role of protein dynamics in the evolution of new enzyme function. Nat Chem Biol 12, 944–950 (2016). https://doi.org/10.1038/nchembio.2175
R.M. Daniel, R.V. Dunn, J.L. Finney, and J.C. Smith, The Role of Dynamics in Enzyme Activity
Annual Review of Biophysics and Biomolecular Structure 2003 32:1, 69-92
Vickery L. Arcus and Adrian J. Mulholland, Temperature, Dynamics, and Enzyme-Catalyzed Reaction Rates
Annual Review of Biophysics 2020 49:1, 163-180
Morgenstern A, Jaszai M, Eberhart ME, Alexandrova AN. Quantified electrostatic preorganization in enzymes using the geometry of the electron charge density. Chem Sci. 2017 Jul 1;8(7):5010-5018. doi: 10.1039/c7sc01301a. Epub 2017 Apr 24. PMID: 28970888; PMCID: PMC5612031.
Andrew J. Adamczyka, Jie Caoa, Shina C. L. Kamerlinb,1, and Arieh Warshela Catalysis by dihydrofolate reductase and other enzymes arises from electrostatic preorganization, not conformational motions
,1www.pnas.org/cgi/doi/10.1073/pnas.1111252108
Li L, Luo M, Ghanem M, Taylor EA, Schramm VL. Second-sphere amino acids contribute to transition-state structure in bovine purine nucleoside phosphorylase. Biochemistry. 2008 Feb 26;47(8):2577-83. doi: 10.1021/bi7021365. PMID: 18281958. PNAS ∣ August 23, 2011 ∣ vol. 108 ∣ no. 34 ∣ 14115–14120
Birktoft JJ, Banaszak LJ. The presence of a histidine-aspartic acid pair in the active site of 2-hydroxyacid dehydrogenases. X-ray refinement of cytoplasmic malate dehydrogenase. J Biol Chem. 1983 Jan 10;258(1):472-82. doi: 10.2210/pdb2mdh/pdb. PMID: 6848515.
Martine Lemaire, Myroslawa Miginiac-Maslow, Paulette Decottignies, The Catalytic Site of Chloroplastic NADP-Dependent Malate Dehydrogenase Contains A His/Asp Pair., European Journal of Biochemistry
March 1996. https://doi.org/10.1111/j.1432-1033.1996.00947.x
Ekici, Ö.D., Paetzel, M. and Dalbey, R.E. (2008), Unconventional serine proteases: Variations on the catalytic Ser/His/Asp triad configuration. Protein Science, 17: 2023-2037. https://doi.org/10.1110/ps.035436.108
Sophonie Jean, & Ellis Bell., Defining the Role of “Second Sphere” Residues in the Activity of Glyoxasomal Malate Dehydrogenase, March 2006., https://doi.org/10.1096/fasebj.20.4.A45-d
Bell et al: The Multiple Roles of the Active Site Loop of Watermelon Glyoxysomal Malate Dehydrogenase, 13 May 2022, https://doi.org/10.1096/fasebj.2022.36.S1.R2813
Hannah Blythe, Ellis Bell, Jessica Bell., Evolution and Adaptation in Malate Dehydrogenases, 18 April 2020., https://doi.org/10.1096/fasebj.2020.34.s1.06310
Wazo Myint, Ellis Bell., Crystallographic Studies of Glyoxysomal Malate Dehydrogenase Mutants D193Nand H220Q
First published: 07 March 2006., https://doi.org/10.1096/fasebj.20.5.A907-c
Shannon Hedrick, Ellis Bell., The Effects of Loop Mutations on the Activity and Regulation of MalateDehydrogenase
First published: 01 April 2009
https://doi.org/10.1096/fasebj.23.1_supplement.LB219
3-5 Learning goals for this CURE:
1. Students will appreciate that a good research project entails nine essential elements of research and will develop a novel hypothesis that makes predictions that can be tested experimentally, and present a proposal for their project. Rubric 1
2. Students will learn how to design and execute experiments to test their hypothesis, will learn appropriate data analysis approaches and will appreciate the importance of accurate documentation of their work and reproducibility of their experiments. Rubric 2
3. Students will learn to develop a description of their research project in written, poster or a slide presentation suitable for verbal presentation. Rubric 3
Research question for this CURE:
1. Can you explore the role of loop dynamics in the catalytic steps of the reaction?
2. Can you understand the structure-function relationships of the protein dynamics that underpin substrate specificity?
3. Can you propose and initiate potential strategies for altering the specificity of the enzyme for biotech purposes?
2-3 Sample hypotheses students could come up with for this CURE:
Introductory lectures have defined types of approaches to drug development and can be broken down to two basic approaches: structure based drug design or a screening potential candidates approach. Students can select one of these approaches or the approach can be set by the instructor.
STUDENTS ARE LED THROUGH HYPOTHESIS AND PROPOSAL PREPARATION USING THE RULE OF THREES APPROACH:
Typically student hypotheses hone in on some unique aspect of theP falci MDH protein structure (established by Clustal Analysis and computational analysis), or some unique aspect of an actual or a potential ligand depending on the overall approach they choose to take (structure based ligand design or screening approach)
What Experimental Approaches can be Incorporated?
WET LAB TECHNIQUES: LINK TO THEORY AND PRACTICAL DESCRIPTIONS OF WET LAB TECHNIQUES
Computational Techniques
CURE FORMAT: MODULAR, SEMESTER, EITHER
Week by week lab activities for a modular version (6 weeks) and/or semester long version of this CURE :
The CURE starts with discussions of the target enzyme, Malate Dehydrogenase and its roles in metabolism. This is followed by details of the reaction catalyzed and the role that dynamic aspects of protein structure might play in aspects of specificity and catalysis. After appropriate literature review and bioinformatics student groups decide on a relevance angle and what basic science issue they want to explore, focusing on either the role of protein dynamics or the roles of “second sphere” residues in catalysis of ligand specificity. Students develop a hypothesis, and as appropriate make the appropriate mutants (or select from existing mutants), express and purify proteins (mutant and wild type) and conduct the various experiments they propose to explore their hypothesis.
Ideal group size for this CURE: Groups of 2-3 students
Ideal course/level for this CURE (chem, bio, biochem, interdisciplinary; first year, middle years, capstone): (list as many as are possibilities)
middle years, upper level
Teaching Resources Available:
Powerpoints to Introduce Each Weeks Lab Session
Generic Customizable Templates for student activities for each of the 9 essential elements of research incorporated into the CURE
T4: Proposal
T5: Experimental Design & Execution
T6: Reproducibility
T7: Data Analysis & Conclusions
Rubrics to Guide & Assess Student Performance
Instrumentation/equipment/key reagents needed for this CURE
Uv-vis Spectrophotometer, Equipment for acid-base titrations, pH Meter, Balance, Water Bath, Stir Plate
Protein (WT and/or specific mutant), organism:
Plasmids needed can be obtained from Addgene:
Plasmodium falciparum, Human Mitochondrial, Human Cytosolic
Clone Data Sheets for this Project Area: While many students choose to work with watermelon glyoxysomal MDH, in reality any of the wild type isoforms available could be used.
Watermelon Glyoxysomal Malate Dehydrogenase
Ignicoccus Islandicus Malate Dehydrognease
MW(subunit/biological)/pI/ e280 , extinction coefficient (280 nm: calculated using ProtParam.) of protein (WT and/or specific mutant):
Plasmodium falciparum: MWt: 35,715/142,860, pI(theoretical): 6.89 e280 0.375 mL.mg-1.cm-1
Human Mitochondrial: MWt: 34,806/69612, pI(theoretical): 8.33 e280 0.257 mL.mg-1.cm-1
Human Cytosolic:MWt: 39,749/79,498, pI(theoretical): 7.14 e280 0.853 mL.mg-1.cm-1
PDB ID for the WT version of these protein :
Plasmodium falciparum: 5.nfr.pdb
Human Cytosolic: 7rm9.pdb
Human Mitochondrial: 2dfd.pdb
Available Resources for structural analysis and computational approaches: Biologically relevant pdb files
Plasmodium falciparum Tetramer
Human Cytosolic Dimer
Human Mitochondrial Dimer
Landmarked .pse files for use with the project - see clone datasheets for descriptions