5tsu
Active conformation for Engineered human cystathionine gamma lyase (E59N, R119L, E339V) to depleting methionine
Structural highlights
DiseaseCGL_HUMAN Defects in CTH are the cause of cystathioninuria (CSTNU) [MIM:219500. It is an autosomal recessive phenotype characterized by abnormal accumulation of plasma cystathionine, leading to increased urinary excretion.[1] [2] FunctionCGL_HUMAN Catalyzes the last step in the trans-sulfuration pathway from methionine to cysteine. Has broad substrate specificity. Converts cystathionine to cysteine, ammonia and 2-oxobutanoate. Converts two cysteine molecules to lanthionine and hydrogen sulfide. Can also accept homocysteine as substrate. Specificity depends on the levels of the endogenous substrates. Generates the endogenous signaling molecule hydrogen sulfide (H2S), and so contributes to the regulation of blood pressure. Acts as a cysteine-protein sulfhydrase by mediating sulfhydration of target proteins: sulfhydration consists of converting -SH groups into -SSH on specific cysteine residues of target proteins such as GAPDH, PTPN1 and NF-kappa-B subunit RELA, thereby regulating their function.[3] [4] [5] Publication Abstract from PubMedEnzyme 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-gamma-lyase scaffold (hCGL-NLV) to circumvent immunogenicity and stability issues observed in the preclinical application of bacterially derived methionine-gamma-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 A) 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 beta- and gamma-eliminations. Our study suggests that solvent ions such as NaCl disrupt electrostatic interactions between R62 and PLP, decreasing catalytic efficiency. Structural Snapshots of an Engineered Cystathionine-gamma-lyase Reveal the Critical Role of Electrostatic Interactions in the Active Site.,Yan W, Stone E, Zhang YJ Biochemistry. 2017 Feb 14;56(6):876-885. doi: 10.1021/acs.biochem.6b01172. Epub, 2017 Feb 3. PMID:28106980[6] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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