title: A sucrose-binding site provides a lead towards an isoform-specific inhibitor of the cancer-associated enzyme carbonic anhydrase IX.
abstract: Human carbonic anhydrase (CA; EC 4.2.1.1) isoform IX (CA IX) is an extracellular zinc metalloenzyme that catalyzes the reversible hydration of CO2 to HCO3(-), thereby playing a role in pH regulation. The majority of normal functioning cells exhibit low-level expression of CA IX. However, in cancer cells CA IX is upregulated as a consequence of a metabolic transition known as the Warburg effect. The upregulation of CA IX for cancer progression has drawn interest in it being a potential therapeutic target. CA IX is a transmembrane protein, and its purification, yield and crystallization have proven challenging to structure-based drug design, whereas the closely related cytosolic soluble isoform CA II can be expressed and crystallized with ease. Therefore, we have utilized structural alignments and site-directed mutagenesis to engineer a CA II that mimics the active site of CA IX. In this paper, the X-ray crystal structure of this CA IX mimic in complex with sucrose is presented and has been refined to a resolution of 1.5 Å, an Rcryst of 18.0% and an Rfree of 21.2%. The binding of sucrose at the entrance to the active site of the CA IX mimic, and not CA II, in a non-inhibitory mechanism provides a novel carbohydrate moiety binding site that could be further exploited to design isoform-specific inhibitors of CA IX.
authors.affiliation: Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville, FL 32610, USA.
authors.forename: Melissa A
authors.initials: MA
authors.lastname: Pinard
authors.affiliation: Division of Biology and Soft Matter, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA.
authors.forename: Mayank
authors.initials: M
authors.lastname: Aggarwal
authors.affiliation: Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville, FL 32610, USA.
authors.forename: Brian P
authors.initials: BP
authors.lastname: Mahon
authors.affiliation: Department of Pharmacology, College of Medicine, University of Florida, Gainesville, FL 32610, USA.
authors.forename: Chingkuang
authors.initials: C
authors.lastname: Tu
authors.affiliation: Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville, FL 32610, USA.
authors.forename: Robert
authors.initials: R
authors.lastname: McKenna
mesh_terms: D000951:Antigens, Neoplasm / Q000378:metabolism*; D001665:Binding Sites; D000071231:Carbonic Anhydrase IX; D002257:Carbonic Anhydrase Inhibitors / Q000737:chemistry* / Q000494:pharmacology*; D002256:Carbonic Anhydrases / Q000378:metabolism*; D003460:Crystallization; D018360:Crystallography, X-Ray; D005470:Fluorometry; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism; D007700:Kinetics; D009369:Neoplasms / Q000201:enzymology*; D013395:Sucrose / Q000378:metabolism*
chemical_list: D000951:Antigens, Neoplasm; D002257:Carbonic Anhydrase Inhibitors; D007527:Isoenzymes; D013395:Sucrose; C089226:CA9 protein, human; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases
keywords: CA IX mimic; carbonic anhydrase IX; isoform-specific drug design; sucrose; sugar approach; α-carbonic anhydrase