Crystal structure of salt-tolerant glutaminase from Micrococcus luteus K-3 in the presence and absence of its product L-glutamate and its activator Tris.
Glutaminase from Micrococcus luteus K-3 [Micrococcus glutaminase (Mglu); 456 amino acid residues (aa); 48 kDa] is a salt-tolerant enzyme. Our previous study determined the structure of its major 42-kDa fragment. Here, using new crystallization conditions, we determined the structures of the intact enzyme in the presence and absence of its product L-glutamate and its activator Tris, which activates the enzyme by sixfold. With the exception of a 'lid' part (26-29 aa) and a few other short stretches, the structures were all very similar over the entire polypeptide chain. However, the presence of the ligands significantly reduced the length of the disordered regions: 41 aa in the unliganded structure (N), 21 aa for L-glutamate (G), 8 aa for Tris (T) and 6 aa for both L-glutamate and Tris (TG). L-glutamate was identified in both the G and TG structures, whereas Tris was only identified in the TG structure. Comparison of the glutamate-binding site between Mglu and salt-labile glutaminase (YbgJ) from Bacillus subtilis showed significantly smaller structural changes of the protein part in Mglu. A comparison of the substrate-binding pocket of Mglu, which is highly specific for L-glutamine, with that of Erwinia carotovora asparaginase, which has substrates other than L-glutamine, shows that Mglu has a larger substrate-binding pocket that prevents the binding of L-asparagine with proper interactions.
Yoshimune
Kazuaki
K
Research Institute of Genome-based Biofactory, National Institute of Advanced Industrial Science and Technology (AIST), Sapporo, Hokkaido, Japan. k.yoshimune@aist.go.jp
Shirakihara
Yasuo
Y
Wakayama
Mamoru
M
Yumoto
Isao
I
D001665:Binding Sites; D020134:Catalytic Domain; D003460:Crystallization; D018360:Crystallography, X-Ray; D018698:Glutamic Acid / Q000378:metabolism*; D005972:Glutaminase / Q000737:chemistry*; D005973:Glutamine / Q000378:metabolism; D016982:Micrococcus luteus / Q000201:enzymology; D014325:Tromethamine / Q000494:pharmacology*
D014325:Tromethamine; D005973:Glutamine; D018698:Glutamic Acid; D005972:Glutaminase; C477762:salt-tolerant glutaminase