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Structure and Function of Metallohydrolases in the Arginase- Deacetylase Family

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Structure and Function of Metallohydrolases in the Arginase- Deacetylase Family University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2016 Structure and Function of Metallohydrolases in the Arginase- Deacetylase Family Yang Hai University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Biochemistry Commons Recommended Citation Hai, Yang, "Structure and Function of Metallohydrolases in the Arginase-Deacetylase Family" (2016). Publicly Accessible Penn Dissertations. 1753. https://repository.upenn.edu/edissertations/1753 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/1753 For more information, please contact [email protected]. Structure and Function of Metallohydrolases in the Arginase-Deacetylase Family Abstract Arginases and deacetylases are metallohydrolases that catalyze two distinct chemical transformations. The arginases catalyze the hydrolysis of the guanidinium group of arginine by using a hydroxide ion 2+ 2+ bridging the binuclear manganese cluster (Mn A-Mn B) for nucleophilic attack. The deacetylases catalyze the hydrolysis of amide bonds by using a mononuclear Zn2+-ion activated water molecule as the nucleophile. Despite the diverse functions, metallohydrolases of the arginase-deacetylase superfamily 2+ share the same characteristic α/β hydrolase core fold and a conserved metal binding site (the Mn B site in arginase corresponds to the catalytic Zn2+ site in deacetylase) which is essential for catalysis in both enzymes. We report crystal structure of formiminoglutamase from the parasitic protozoan Trypanosoma cruzi and confirm that formiminoglutamase is a Mn2+-requiring hydrolase that belongs to the arginase- deacetylase superfamily. We also report the crystal structure of an arginase-like protein from Trypanosoma brucei (TbARG) with unknown function. Although its biological role remains enigmatic, the 2+ evolutionarily more conserved Mn B site can be readily restored in TbARG through side-directed mutagenesis. We also report crystal structure of an arginase from the parasite Schistosoma mansoni (SmARG). Structural comparison of SmARG complexed with second-generation arginase inhibitors (α,α- disubstituted boronic acid inhibitors) with another parasitic arginase from Leishmania mexicana and human arginases reveal interesting differences in the binding modes of the additional α-substituents. Reversible lysine acetylation rivals phosphorylation in the regulation of protein structure and function, and inhibition of the “eraser” histone deacetylase (HDAC) is a validated approach for cancer chemotherapy. HDAC6, the sole HDAC that harbors a full duplication of catalytic domain (CD1 and CD2), is a cytosolic lysine deacetylase known to deacetylate α-tubulin, heat-shock protein 90, etc. Here, we report HDAC6 structures that provide new insights about mechanism, catalysis, and inhibitor binding. In light of biochemical studies, we reveal a “gate constriction” mechanism responsible for the strict substrate specificity of CD1 versus broad substrate specificity of CD2. Analysis of other isozymes indicates that the closest relative HDAC10 contains an alternative gatekeeper that favors catalysis with acetylpolyamines. Indeed, we provide structural evidence that HDAC10 is the long-sought mammalian N8-acetylspermidine deacetylase whose identity has remained elusive for 30 years. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Chemistry First Advisor David W. Christianson Keywords Arginase, Crystallography, Histone deacetylase, Metallohydrolase, polyamine deacetylase Subject Categories Biochemistry This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/1753 STRUCTURE AND FUNCTION OF METALLOHYDROLASES IN THE ARGINASE- DEACETYLASE FAMILY Yang Hai A DISSERTATION in Chemistry Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2016 Supervisor of Dissertation ________________________ Dr. David W. Christianson Roy and Diana Vagelos Professor in Chemistry and Chemical Biology Graduate Group Chairperson ________________________ Dr. Gary A. Molander Hirschmann-Makineni Professor of Chemistry Dissertation Committee Dr. Ronen Marmorstein, Professor of Chemistry, Biochemistry and Molecular Biology Dr. Barry S. Cooperman, Professor of Chemistry Dr. Ivan J. Dmochowski, Professor of Chemistry ACKNOWLEDGMENT I am deeply indebted to my thesis advisor, Prof. David Christianson, who has always been an amazing mentor to me, both personally and professionally. This dissertation would not have been possible without him giving me the opportunity at first time to work in his lab four years ago, without his guidance throughout my thesis study in the past four years, without him giving me such freedom to think independently and freely, and allowing me to work in such a flexible way that I can have the privilege to study all kinds of subjects that we both are interested in. I sincerely thank my thesis committee members: Prof. Ronen Marmorstein, Prof. Ivan Dmochowski, and Prof. Barry Cooperman. I am so grateful for having these excellent faculty members sitting on my committee and I really appreciate their time and their many suggestions which help to shape this thesis to what it is today. Particularly, I would like to thank Prof. Ronen Marmorstein for his many valuable insightful advice and discussions, and for him including me in the Crystal Talk Community. I also greatly appreciate the support from members of Christianson lab and value the friendship with them, including but not limited to Dr. Edward D’Antonio, Dr. Mustafa Köksal, Dr. Mo Chen, Dr. Ruiqiong Li, Dr. Christophe Decroos, Mengbin Chen, Alex Genshaft, Reilly Dugery, In particular, I would like to thank Dr. Edward D’Antonio, who introduced me onto his so-called “Crystal Road”; as well as Dr. Mustafa Köksal, who is always my role model in the lab ever since. I would also like to thank our good neighbors–Chenoweth lab–for allowing me to get acess to their many instruments. Especially, I would like to thank my close friend Yitao Zhang for his countless help and Prof. David Chenoweth for his much advice. Last but not least, I would like to thank my parents, my family, my friends in China, and my friends from graduate school who I haven’t mentioned yet. I could not have made it this far without your love, support, and encouragement. I really do owe you all so much. Thank you, Penn, for leaving me such a good memory. Thank you for all the good and bad times. ii ABSTRACT STRUCTURE AND FUNCTION OF METALLOHYDROLASES IN THE ARGINASE- DEACETYLASE FAMILY Yang Hai David W. Christianson Arginases and deacetylases are metallohydrolases that catalyze two distinct chemical transformations. The arginases catalyze the hydrolysis of the guanidinium group of arginine by 2+ 2+ using a hydroxide ion bridging the binuclear manganese cluster (Mn A-Mn B) for nucleophilic attack. The deacetylases catalyze the hydrolysis of amide bonds by using a mononuclear Zn2+- ion activated water molecule as the nucleophile. Despite the diverse functions, metallohydrolases of the arginase-deacetylase superfamily share the same characteristic α/β hydrolase core fold 2+ 2+ and a conserved metal binding site (the Mn B site in arginase corresponds to the catalytic Zn site in deacetylase) which is essential for catalysis in both enzymes. We report crystal structure of formiminoglutamase from the parasitic protozoan Trypanosoma cruzi and confirm that formiminoglutamase is a Mn2+-requiring hydrolase that belongs to the arginase-deacetylase superfamily. We also report the crystal structure of an arginase-like protein from Trypanosoma brucei (TbARG) with unknown function. Although its 2+ biological role remains enigmatic, the evolutionarily more conserved Mn B site can be readily restored in TbARG through side-directed mutagenesis. We also report crystal structure of an arginase from the parasite Schistosoma mansoni (SmARG). Structural comparison of SmARG complexed with second-generation arginase inhibitors (α,α-disubstituted boronic acid inhibitors) with another parasitic arginase from Leishmania mexicana and human arginases reveal interesting differences in the binding modes of the additional α-substituents. iii Reversible lysine acetylation rivals phosphorylation in the regulation of protein structure and function, and inhibition of the “eraser” histone deacetylase (HDAC) is a validated approach for cancer chemotherapy. HDAC6, the sole HDAC that harbors a full duplication of catalytic domain (CD1 and CD2), is a cytosolic lysine deacetylase known to deacetylate α-tubulin, heat- shock protein 90, etc. Here, we report HDAC6 structures that provide new insights about mechanism, catalysis, and inhibitor binding. In light of biochemical studies, we reveal a “gate constriction” mechanism responsible for the strict substrate specificity of CD1 versus broad substrate specificity of CD2. Analysis of other isozymes indicates that the closest relative HDAC10 contains an alternative gatekeeper that favors catalysis with acetylpolyamines. Indeed, we provide structural evidence that HDAC10 is the long-sought mammalian N8-acetylspermidine deacetylase whose identity has remained elusive for 30 years. iv TABLE OF CONTENTS Acknowledgement….………………………..……………………...……………………………………ii Abstract…….…...…..………………………..………………………...…………………………………iii Table of contents……..……….………..………………………………...….……………...……………v List of Tables…..……….………..…………………...……………………………...……...…………..vii
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