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Title:Structure, function and inhibition of GcpE, FPPS and GGPPS: targeting isoprenoid biosynthesis for drug discovery
Author(s):Liu, Yi-Liang
Director of Research:Oldfield, Eric
Doctoral Committee Chair(s):Oldfield, Eric
Doctoral Committee Member(s):Gennis, Robert B.; Mitchell, Douglas A.; Nair, Satish K.
Department / Program:School of Molecular & Cell Bio
Discipline:Biophysics & Computnl Biology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):X-ray Crystallography
Iron-sulfur protein
Catalysis
Drug Discovery
IspG
GcpE
geranylgeranyl diphosphate synthase (GGPPS)
farnesyl diphosphate synthase (FPPS)
Abstract:IspG, farnesyl diphosphate synthase (FPPS) and geranylgeranyl diphosphate synthase (GGPPS) are enzymes involved in isoprenoid biosynthesis. Most bacterial IspGs contain two domains: a TIM barrel (A) and a 4Fe4S domain (B), but in plants and malaria parasites there is a large insert domain (A*) whose structure and function are unknown. We show bacterial IspGs function in solution as (AB)2 dimers and mutations in either both A or both B domains block activity. Chimeras harboring an A-mutation in one chain and a B-mutation in the other have 50% of the activity seen in wild-type protein, since there is still one catalytically active AB domain. However, a plant IspG functions as an AA*B monomer and we propose using computational modeling and electron microscopy that the A* insert domain has a TIM barrel structure that interacts with the A domain. This structural arrangement enables the A and B domains to interact in a “cup and ball” manner during catalysis, just as in the bacterial systems. EPR/HYSCORE spectra of reaction intermediate, product and inhibitor ligands bound to both two and three domain proteins are identical, indicating the same local electronic structure, and computational docking indicates these ligands bridge both A and B domains. Overall, the results are of broad general interest since they indicate the insert domain in three-domain IspGs is a second TIM barrel that plays a structural role and that the pattern of inhibition of both two and three domain proteins are the same, results that can be expected to be of use in drug design. We have found geranylgeranyl diphosphate synthase (GGPPS), a dual-functional enzyme that produces farnesyl diphosphate and geranylgeranyl diphosphate appears to be a good target. Lipophilic bisphosphonates as well as a new class of inhibitor, benzoic acids, have good inhibition (Ki~1μM) against Plasmodium vivax GGPPS. Benzoic acid analogs have been synthesized and tested against PvGGPPS. Crystal structures of several lipophilic bisphosphonates as well as benzoic acid derivatives bound to PvGGPPS have been solved. The lipophilic bisphosphonate (BPH-703) binds to the FPP site, but all benzoic acids bind to the IPP site. Human FPPS has been proved to be a validated drug target for osteoporosis-related diseases and cancers. We found that the BPH-703 and its analogs have an effect on chain length dependence against HsFPPS and PvGGPPS enzymes as well as cells (Plasmodium parasites), in vitro. Activation of γδ T cell experiment shows similar effects on chain length variation. Crystal structure of HsFPPS bound to BPH-1260, a C4 version of zoledronate with a hydroxyl group on the bisphosphonate group explains the intrinsic properties of chain length dependence of BPH-703 derivatives. Making potent and selective inhibitors against these three enzymes are important as anti-cancer and anti-infective agents.
Issue Date:2013-02-03
URI:http://hdl.handle.net/2142/42471
Rights Information:Copyright 2012 Yi-Liang Liu
Date Available in IDEALS:2013-02-03
2015-02-03
Date Deposited:2012-12


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