Abstract:
:The members of the glutamine amidotransferase (GATase) family catalyze the incorporation of ammonia within numerous metabolic pathways and can be categorized in two classes. Here, we concentrated on class I GATases, which are heteromeric enzyme complexes consisting of synthase subunits and glutaminase subunits with a catalytic Cys-His-Glu triad. Glutamine hydrolysis at the glutaminase subunit is (i) dependent on the formation of tight synthase-glutaminase complexes and (ii) allosterically coupled to the presence of the substrate at the synthase subunit. The structural basis of both complex formation and allostery is poorly understood. However, previous work on 4-amino-4-deoxychorismate synthase and imidazole glycerol phosphate synthase suggested that a conserved aspartate residue in their synthase subunits, which is located at the subunit interface close to the glutaminase catalytic triad, might be important for both features. We performed a computational screen of class I GATases from the Protein Data Bank and identified conserved and similarly located aspartate residues. We then generated alanine and glutamate mutants of these residues and characterized them by analytical gel filtration and steady-state enzyme kinetics. The results confirmed the important role of the wild-type aspartate residues for the formation of stable synthase-glutaminase complexes (in three of four cases) and the stimulation of glutaminase activity in the analyzed GATases (in all four cases). We present a model for rationalizing the dual role of the conserved aspartate residue toward a unifying regulation mechanism in the entire class I GATase family.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Semmelmann F,Hupfeld E,Heizinger L,Merkl R,Sterner Rdoi
10.1021/acs.biochem.9b00286subject
Has Abstractpub_date
2019-06-04 00:00:00pages
2584-2588issue
22eissn
0006-2960issn
1520-4995journal_volume
58pub_type
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