4HG5

Structural insights into yeast Nit2: wild-type yeast Nit2 in complex with oxaloacetate

Summary for 4HG5

• Entry DOI: 10.2210/pdb4hg5/pdb
• Related: 4H5U 4HG3 4HGD
• Descriptor: Probable hydrolase NIT2, OXALOACETATE ION, GLYCEROL, ... (5 entities in total)
• Functional Keywords: nitrilase superfamily, omega-amidase, hydrolase
• Biological source: Saccharomyces cerevisiae (Baker's yeast)
• Total number of polymer chains: 4
• Total formula weight: 154802.32

Authors

Liu, H.,Qiu, X.,Zhang, M.,Gao, Y.,Niu, L.,Teng, M. (deposition date: 2012-10-07, release date: 2013-07-31, Last modification date: 2024-10-16)

Primary citation

Liu, H.,Gao, Y.,Zhang, M.,Qiu, X.,Cooper, A.J.L.,Niu, L.,Teng, M.
Structures of enzyme-intermediate complexes of yeast Nit2: insights into its catalytic mechanism and different substrate specificity compared with mammalian Nit2
Acta Crystallogr.,Sect.D, 69:1470-1481, 2013

PubMed Abstract: The Nit (nitrilase-like) protein subfamily constitutes branch 10 of the nitrilase superfamily. Nit proteins are widely distributed in nature. Mammals possess two members of the Nit subfamily, namely Nit1 and Nit2. Based on sequence similarity, yeast Nit2 (yNit2) is a homologue of mouse Nit1, a tumour-suppressor protein whose substrate specificity is not yet known. Previous studies have shown that mammalian Nit2 (also a putative tumour suppressor) is identical to ω-amidase, an enzyme that catalyzes the hydrolysis of α-ketoglutaramate (α-KGM) and α-ketosuccinamate (α-KSM) to α-ketoglutarate (α-KG) and oxaloacetate (OA), respectively. In the present study, crystal structures of wild-type (WT) yNit2 and of WT yNit2 in complex with α-KG and with OA were determined. In addition, the crystal structure of the C169S mutant of yNit2 (yNit2-C169S) in complex with an endogenous molecule of unknown structure was also solved. Analysis of the structures revealed that α-KG and OA are covalently bound to Cys169 by the formation of a thioester bond between the sulfhydryl group of the cysteine residue and the γ-carboxyl group of α-KG or the β-carboxyl group of OA, reflecting the presumed reaction intermediates. However, an enzymatic assay suggests that α-KGM is a relatively poor substrate of yNit2. Finally, a ligand was found in the active site of yNit2-C169S that may be a natural substrate of yNit2 or an endogenous regulator of enzyme activity. These crystallographic analyses provide information on the mode of substrate/ligand binding at the active site of yNit2 and insights into the catalytic mechanism. These findings suggest that yNit2 may have broad biological roles in yeast, especially in regard to nitrogen homeostasis, and provide a framework for the elucidation of the substrate specificity and biological role of mammalian Nit1.

PubMed: 23897470
DOI: 10.1107/S0907444913009347

Experimental method

X-RAY DIFFRACTION (1.91 Å)