title: Structural Snapshots of an Engineered Cystathionine-γ-lyase Reveal the Critical Role of Electrostatic Interactions in the Active Site.
abstract: Enzyme therapeutics that can degrade l-methionine (l-Met) are of great interest as numerous malignancies are exquisitely sensitive to l-Met depletion. To exhaust the pool of methionine in human serum, we previously engineered an l-Met-degrading enzyme based on the human cystathionine-γ-lyase scaffold (hCGL-NLV) to circumvent immunogenicity and stability issues observed in the preclinical application of bacterially derived methionine-γ-lyases. To gain further insights into the structure-activity relationships governing the chemistry of the hCGL-NLV lead molecule, we undertook a biophysical characterization campaign that captured crystal structures (2.2 Å) of hCGL-NLV with distinct reaction intermediates, including internal aldimine, substrate-bound, gem-diamine, and external aldimine forms. Curiously, an alternate form of hCGL-NLV that crystallized under higher-salt conditions revealed a locally unfolded active site, correlating with inhibition of activity as a function of ionic strength. Subsequent mutational and kinetic experiments pinpointed that a salt bridge between the phosphate of the essential cofactor pyridoxal 5'-phosphate (PLP) and residue R62 plays an important role in catalyzing β- and γ-eliminations. Our study suggests that solvent ions such as NaCl disrupt electrostatic interactions between R62 and PLP, decreasing catalytic efficiency.
authors.affiliation: Department of Molecular Biosciences and ‡Institute for Cellular and Molecular Biology, The University of Texas at Austin , Austin, Texas 78712, United States.
authors.forename: Wupeng
authors.initials: W
authors.lastname: Yan
authors.affiliation: Department of Molecular Biosciences and ‡Institute for Cellular and Molecular Biology, The University of Texas at Austin , Austin, Texas 78712, United States.
authors.forename: Everett
authors.identifier: 0000-0002-2459-9762
authors.initials: E
authors.lastname: Stone
authors.affiliation: Department of Molecular Biosciences and ‡Institute for Cellular and Molecular Biology, The University of Texas at Austin , Austin, Texas 78712, United States.
authors.forename: Yan Jessie
authors.identifier: 0000-0002-9360-5388
authors.initials: YJ
authors.lastname: Zhang
mesh_terms: D019943:Amino Acid Substitution; D001120:Arginine / Q000737:chemistry; D055162:Biocatalysis; D013437:Carbon-Sulfur Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D003540:Cystathionine / Q000378:metabolism; D003542:Cystathionine gamma-Lyase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism; D004795:Enzyme Stability; D006801:Humans; D006860:Hydrogen Bonding; D006868:Hydrolysis; D008715:Methionine / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009994:Osmolar Concentration; D011487:Protein Conformation; D015202:Protein Engineering; D011732:Pyridoxal Phosphate / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012645:Selenomethionine / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D011994:Recombinant Proteins; D003540:Cystathionine; D011732:Pyridoxal Phosphate; D001120:Arginine; D012645:Selenomethionine; D008715:Methionine; D013437:Carbon-Sulfur Lyases; D003542:Cystathionine gamma-Lyase; C021558:L-methionine gamma-lyase; D003545:Cysteine