5TT2

Inactive conformation of engineered human cystathionine gamma lyase (E59N, R119L, E339V) to depleting methionine

Summary for 5TT2

• Entry DOI: 10.2210/pdb5tt2/pdb
• Descriptor: Cystathionine gamma-lyase, SULFATE ION (2 entities in total)
• Functional Keywords: engineered protein, methioninase, inactive form, lyase
• Biological source: Homo sapiens (Human)
• Total number of polymer chains: 2
• Total formula weight: 93604.46

Authors

Yan, W.,Zhang, Y. (deposition date: 2016-10-31, release date: 2017-10-11, Last modification date: 2025-04-02)

Primary citation

Yan, W.,Stone, E.,Zhang, Y.J.
Structural Snapshots of an Engineered Cystathionine-gamma-lyase Reveal the Critical Role of Electrostatic Interactions in the Active Site.
Biochemistry, 56:876-885, 2017

PubMed 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.

PubMed: 28106980
DOI: 10.1021/acs.biochem.6b01172

Experimental method

X-RAY DIFFRACTION (2.949 Å)