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CATALYTIC MECHANISM and FUNCTION EVOLVEMENT STUDIES of PHOSPHATASES WITHIN HALOACID DEHALOGENASE SUPERFAMILY (HADSF) Li Zheng

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CATALYTIC MECHANISM and FUNCTION EVOLVEMENT STUDIES of PHOSPHATASES WITHIN HALOACID DEHALOGENASE SUPERFAMILY (HADSF) Li Zheng University of New Mexico UNM Digital Repository Chemistry ETDs Electronic Theses and Dissertations 7-10-2013 CATALYTIC MECHANISM AND FUNCTION EVOLVEMENT STUDIES OF PHOSPHATASES WITHIN HALOACID DEHALOGENASE SUPERFAMILY (HADSF) Li Zheng Follow this and additional works at: https://digitalrepository.unm.edu/chem_etds Recommended Citation Zheng, Li. "CATALYTIC MECHANISM AND FUNCTION EVOLVEMENT STUDIES OF PHOSPHATASES WITHIN HALOACID DEHALOGENASE SUPERFAMILY (HADSF)." (2013). https://digitalrepository.unm.edu/chem_etds/31 This Dissertation is brought to you for free and open access by the Electronic Theses and Dissertations at UNM Digital Repository. It has been accepted for inclusion in Chemistry ETDs by an authorized administrator of UNM Digital Repository. For more information, please contact [email protected]. Li Zheng Candidate Chemistry and Chemical Biology Department This dissertation is approved, and it is acceptable in quality and form for publication: Approved by the Dissertation Committee: Debra Dunaway-Mariano , Chairperson Patrick S. Mariano Wei Wang Fu-Sen Liang Karen N. Allen CATALYTIC MECHANISM AND FUNCTION EVOLVEMENT STUDIES OF PHOSPHATASES WITHIN HALOACID DEHALOGENASE SUPERFAMILY (HADSF) by LI ZHENG B.S., Chemistry, Sichuan University, China, 2002 M.S., Chemistry, Sichuan University, China, 2005 DISSERTATION Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Chemistry The University of New Mexico Albuquerque, New Mexico January, 2013 DEDICATION To My parents Shifu Zheng and Changxiu Pang and My husband Min Wang I ACKNOWLEDGEMENTS I would like to express my deepest appreciation and gratitude to my research advisors Professor Patrick, S. Mariano and Professor Debra Dunaway-Mariano, not only for their scientific advice but also for their personal guidance. Their invaluable advice and everyday encouragement make all these years enjoyable and memorable. Their guidance and professional style will remain with me as I continue my career. Without them I could have never gone so far. They have the respect from the bottom of my heart. I thank my committee members, Dr. Wei Wang, Dr. Karen N. Allen and Dr. Fu-Sen Liang for their valuable recommendations pertaining to this study and assistance in my professional development. I am so grateful to have worked and collaborated with Dr. Karen N. Allen in Boston University. I am grateful to Dr. Kelly Daughtry for her great efforts in solving the crystal structure of YidA. I also thank Dr. Jeremiah Farelli for the high throughput screen for all the HAD phosphatases. Many thanks go to Dr. Yuriy V. Patskovsky in Albert Einstein College of Medicine for solving the structure of BT2542 phosphatase. I would also like to thank all of the past and present members of Dr. Debra Dunaway- Mariano’s research group for their help and friendship. Finally, I would like to thank my husband, Min Wang who supports me greatly not only on my life, but also on my research. Without his support, friendship, love and understanding, none of this would be possible. II Catalytic Mechanism and Function Evolvement Studies of Phosphatases within Haloacid Dehalogenase Superfamily (HADSF) by Li Zheng B.S., Chemistry, Sichuan University, China, 2002 M.S., Chemistry, Sichuan University, China, 2005 Ph.D., Chemistry, University of New Mexico, USA, 2013 ABSTRACT The haloacid dehalogenase superfamily (HADSF) is one of the largest known enzyme superfamilies, having more than 32,000 nonredundant members. Enzymes in this superfamily catalyze a number of different chemical reactions including dehalogenation, phosphoryl transfer, and hydrolysis of phosphate esters and phosphates. The vast majority of HADSF members are phosphatases, which contain a highly conserved core domain that is constructed to catalyze hydrolysis reactions of phosphorylated sugars or nucleotides by using an active site Asp nucleophile and a Mg2+ cofactor. Most phosphatases possess a cap domain, which in association with the core domain assists active site desolvation and binds the region of the substrate that is displaced by water upon Asp nucleophilic attack at the phosphoryl group. In the studies described in this Dissertation, two nucleotidases (AphA and mdN) were chosen to examine how the core Rossmann fold of HADSF members stabilizes the transition state of phosphoryl transfer III reactions and conserves the stable catalytic scaffold under the pressure of evolutionary changes that expand substrate range. Another phosphatase, YidA, was subjected to studies in order to gain information about how accessory cap domains serve as sophisticated substrate recognition sites. AphA is a nonspecific acid phosphohydrolase (NSAP) from Escherichia coli., which is secreted in the periplasmic space. Although AphA has the highest activity towards 5 -1 -1 hydrolysis of nucleotide phosphates (kcat/Km ~ 10 M s ), it does not yet have an identified biological function. The crystal structures of the apo enzyme as well as complexes of AphA with products of nucleotide monophosphate hydrolysis and with substrate/intermediate analogs have been elucidated. Earlier analysis of these crystal structures shows that a specific hydrophobic substrate binding pocket, formed by Phe56 and Tyr193, exists in AphA. The results of solvent isotope studies carried out in the research effort described in this Dissertation, as well as investigations probing the effect on rates of alcohol additives suggest that the rate-limiting step of the AphA catalyzed hydrolysis reaction of ATP is the second partial reaction in which dephosphorylation of the phosphoenzyme (E-P) intermediate takes place. Observations made in an investigation of the linear free energy relationship between pKa of the alcohol that serves as the nucleophilic phosphoryl acceptor and the second order rate constant for hydrolysis of the E-P intermediate indicate that the mechanism for the rate limiting step of the reaction involving water (or added alcohol) as the nucleophile is highly dissociative. In addition, release of products in this process involves release of inorganic phosphate first followed by release of adenosine. IV mdN is a mitochondrial deoxyribonucleotidase that is believed to function in a salvage pathways to regulate the sizes of deoxyribonucleotide pools. In Chapter 3 of the Dissertation, preliminary results of studies aimed at elucidating the detailed mechanism of mdN catalyzed hydrolysis reactions are presented. The findings show that mdN has activity towards substrates containing aromatic rings like those present in para- nitrophenyl phosphate, nucleotides and deoxynucleotides and that it is not active toward sugar phosphates. Like AphA, mdN contains a hydrophobic substrate-binding pocket formed by residues Phe75, Phe49, Trp76 and Trp96. The results of an investigation of mutli-turnover reactions of 14C dUMP catalyzed by mdN revealed that a pre-steady state burst phase takes place followed by a slow steady-state phase. This observation leads to the conclusion that the substrate-binding step is not the rate-limiting step. YidA is a HADSF phosphatase, isolated from E.coli., that has a C2 cap. This enzyme is an effective catalyst for hydrolysis of several known phosphate metabolites, such as arabinose-5-phosphate, erythrose-4-phosphate, α-mannnose-1-phosphate and α-glucose 1-phosphate as well as two related carboxylic acid derivatives. The YidA gene is located in the same “neighborhood” as the dgo operon, whose genes encode enzymes involved in the galactonate to pyruvate and glyceraldehyde 3-phosphate degradation pathway. Studies described in Chapter 4 show that YidA has a moderate activity toward hydrolysis of the intermediate in this pathway, D-2-dehydro-3-deoxy-D-galactonate-6-phosphate 3 -1 -1 (kcat/Km = 6.4 x 10 M s ). Therefore, YidA is believed to play the role of a housekeeper whose purpose is to remove accumulated phosphorylated metabolites. Studies with a series of site-directed mutants, in which the 10 highly conserved cap residues Arg46, Leu111, His129, Glu130, Lys154, Met156, Ser184, Tyr187, Phe188 and Glu190 are V replaced by Ala, revealed that Glu130, Glu190 and Lys154 play important roles in substrate recognition and binding, while His129 and F188 are critical for binding C6 aldose/carboxylate phosphates. Finally, a program targeted at protein function assignment is described in Chapter 5 of this Dissertation. Protein function assignment has always been a major goal in biology and, as the genome sequence database has rapidly expanded, this task has been significantly magnified. Unfortunately, using of bioinformatics techniques is unsuccessful in assigning the function of between 40-60% of the new enzyme sequences, and the much more demanding experimental methods have been applied to only a tiny fraction of the sequences. Therefore, a more reliable and efficient method for protein function assignment needs to be developed. The Enzyme Function Initiative (EFI), which is a large scale collaborative project that utilizes a multidisciplinary strategy to determine the functions of unknown proteins, was formed based on this consideration. In Chapter 5, the general strategy employed for enzyme functional assignment in this initiative is described along with preliminary experimental results. From the results of high throughput screening, four HADSF phosphatases from different organisms, YigB (EFI-501262) and YbjI(501335) from Escherichia coli, BT2542 (501088) from Bacteroides thetaiotaomicron and Q9RUP0 (501193) from Deinococcus radiodurans, were identified to have
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