Insight on an Arginine Synthesis Metabolon from the Tetrameric Structure of Yeast Acetylglutamate Kinase

Sergio De Cima,Vicente Rubio,Marjolaine Crabeel,Fernando Gil-Ortiz,Ignacio Fita,Inari Kursula

doi:10.1371/journal.pone.0034734

Abstract

N-acetyl-L-glutamate kinase (NAGK) catalyzes the second, generally controlling, step of arginine biosynthesis. In yeasts, NAGK exists either alone or forming a metabolon with N-acetyl-L-glutamate synthase (NAGS), which catalyzes the first step and exists only within the metabolon. Yeast NAGK (yNAGK) has, in addition to the amino acid kinase (AAK) domain found in other NAGKs, a ∼150-residue C-terminal domain of unclear significance belonging to the DUF619 domain family. We deleted this domain, proving that it stabilizes yNAGK, slows catalysis and modulates feed-back inhibition by arginine. We determined the crystal structures of both the DUF619 domain-lacking yNAGK, ligand-free as well as complexed with acetylglutamate or acetylglutamate and arginine, and of complete mature yNAGK. While all other known arginine-inhibitable NAGKs are doughnut-like hexameric trimers of dimers of AAK domains, yNAGK has as central structure a flat tetramer formed by two dimers of AAK domains. These dimers differ from canonical AAK dimers in the −110° rotation of one subunit with respect to the other. In the hexameric enzymes, an N-terminal extension, found in all arginine-inhibitable NAGKs, forms a protruding helix that interlaces the dimers. In yNAGK, however, it conforms a two-helix platform that mediates interdimeric interactions. Arginine appears to freeze an open inactive AAK domain conformation. In the complete yNAGK structure, two pairs of DUF619 domains flank the AAK domain tetramer, providing a mechanism for the DUF619 domain modulatory functions. The DUF619 domain exhibits the histone acetyltransferase fold, resembling the catalytic domain of bacterial NAGS. However, the putative acetyl CoA site is blocked, explaining the lack of NAGS activity of yNAGK. We conclude that the tetrameric architecture is an adaptation to metabolon formation and propose an organization for this metabolon, suggesting that yNAGK may be a good model also for yeast and human NAGSs.

Highlights

Microorganisms and plants make arginine from glutamate using a route in which intermediates are N-acetylated (Figure 1)
This enzyme, which is produced in yeast mitochondria by proteolytic processing of the polyprotein precursor (Uniprot database code Q01217) encoded by the ARG5,6 gene, has the unexplained peculiarity, which is shared by the NAG kinase (NAGK) of other ascomycetes, of having, in addition to the amino acid kinase (AAK) domain, a Cterminal domain of,150 residues of unknown structure and function (Figure 2A) [10,11,12]
The same type of domain is found at the C-terminus in the NAG synthase (NAGS) of ascomycetes and animals, including humans [13,14]. This domain belongs to the DUF619 domain family of the Pfam and InterPro databases. Another very interesting characteristic of Yeast NAGK (yNAGK) is that it forms with yeast NAGS a complex with the features of a true metabolon [12,15], with its NAGS and NAGK components mutually influencing their regulatory properties by arginine [15] (Figure 1)

Summary

Introduction

Microorganisms and plants make arginine from glutamate using a route in which intermediates are N-acetylated (Figure 1). We report here structural and functional studies on the arginine-sensitive NAGK from Saccharomyces cerevisiae (yNAGK) This enzyme, which is produced in yeast mitochondria by proteolytic processing of the polyprotein precursor (Uniprot database code Q01217) encoded by the ARG5,6 gene, has the unexplained peculiarity, which is shared by the NAGKs of other ascomycetes, of having, in addition to the AAK domain, a Cterminal domain of ,150 residues of unknown structure and function (Figure 2A) [10,11,12]. This domain belongs to the DUF619 domain family (where DUF stands for domain of unknown function) of the Pfam (http://pfam.sanger.ac.uk) and InterPro (http://www.ebi.ac.uk/interpro) databases Another very interesting characteristic of yNAGK is that it forms with yeast NAGS (yNAGS) a complex with the features of a true metabolon [12,15], with its NAGS and NAGK components mutually influencing their regulatory properties by arginine [15] (Figure 1). The structure allows to propose a model for the NAGK/NAGS complex forming the yeast metabolon

Results and Discussion

Materials and Methods

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