4HG3: Structural Insights Into Yeast Nit2: Wild-type Yeast Nit2 In Complex With Alpha-ketoglutarate

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 omega-amidase, an enzyme that catalyzes the hydrolysis of alpha-ketoglutaramate (alpha-KGM) and alpha-ketosuccinamate (alpha-KSM) to alpha-ketoglutarate (alpha-KG) and oxaloacetate (OA), respectively. In the present study, crystal structures of wild-type (WT) yNit2 and of WT yNit2 in complex with alpha-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 alpha-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 gamma-carboxyl group of alpha-KG or the beta-carboxyl group of OA, reflecting the presumed reaction intermediates. However, an enzymatic assay suggests that alpha-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.
PDB ID: 4HG3Download
MMDB ID: 112222
PDB Deposition Date: 2012/10/6
Updated in MMDB: 2013/08
Experimental Method:
x-ray diffraction
Resolution: 1.93  Å
Source Organism:
Similar Structures:
Biological Unit for 4HG3: dimeric; determined by author and by software (PISA)
Molecular Components in 4HG3
Label Count Molecule
Proteins (2 molecules)
Probable Hydrolase Nit2(Gene symbol: NIT2)
Molecule annotation
Chemicals (16 molecules)
* Click molecule labels to explore molecular sequence information.

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