Crystal structure of human T-protein of glycine cleavage system at 2.0 A resolution and its implication for understanding non-ketotic hyperglycinemia.
T-protein, a component of the glycine cleavage system, catalyzes the formation of ammonia and 5,10-methylenetetrahydrofolate from the aminomethyl moiety of glycine attached to the lipoate cofactor of H-protein. Several mutations in the human T-protein gene cause non-ketotic hyperglycinemia. To gain insights into the effect of disease-causing mutations and the catalytic mechanism at the molecular level, crystal structures of human T-protein in free form and that bound to 5-methyltetrahydrofolate (5-CH3-H4folate) have been determined at 2.0 A and 2.6 A resolution, respectively. The overall structure consists of three domains arranged in a cloverleaf-like structure with the central cavity, where 5-CH3-H4folate is bound in a kinked shape with the pteridine group deeply buried into the hydrophobic pocket and the glutamyl group pointed to the C-terminal side surface. Most of the disease-related residues cluster around the cavity, forming extensive hydrogen bonding networks. These hydrogen bonding networks are employed in holding not only the folate-binding space but also the positions and the orientations of alpha-helix G and the following loop in the middle region, which seems to play a pivotal role in the T-protein catalysis. Structural and mutational analyses demonstrated that Arg292 interacts through water molecules with the folate polyglutamate tail, and that the invariant Asp101, located close to the N10 group of 5-CH3-H4folate, might play a key role in the initiation of the catalysis by increasing the nucleophilic character of the N10 atom of the folate substrate for the nucleophilic attack on the aminomethyl lipoate intermediate. A clever mechanism of recruiting the aminomethyl lipoate arm to the reaction site seems to function as a way of avoiding the release of toxic formaldehyde.
Okamura-Ikeda
Kazuko
K
Institute for Enzyme Research, The University of Tokushima, Tokushima 770-8503, Japan. ikeda@ier.tokushima-u.ac.jp
Hosaka
Harumi
H
Yoshimura
Masato
M
Yamashita
Eiki
E
Toma
Sachiko
S
Nakagawa
Atsushi
A
Fujiwara
Kazuko
K
Motokawa
Yutaro
Y
Taniguchi
Hisaaki
H
D000595:Amino Acid Sequence; D050960:Aminomethyltransferase; D000818:Animals; D001120:Arginine / Q000737:chemistry; D001216:Asparagine / Q000737:chemistry; D001665:Binding Sites; D020134:Catalytic Domain; D016000:Cluster Analysis; D018360:Crystallography, X-Ray / Q000379:methods*; D004252:DNA Mutational Analysis; D005557:Formaldehyde / Q000737:chemistry; D005998:Glycine / Q000737:chemistry*; D006801:Humans; D006860:Hydrogen Bonding; D019877:Hydroxymethyl and Formyl Transferases / Q000737:chemistry*; D020158:Hyperglycinemia, Nonketotic / Q000378:metabolism*; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid
D005557:Formaldehyde; D001216:Asparagine; D001120:Arginine; D019877:Hydroxymethyl and Formyl Transferases; D050960:Aminomethyltransferase; D005998:Glycine