A Fold-Independent Interface Residue Is Crucial for Complex Formation and Allosteric Signaling in Class I Glutamine Amidotransferases.

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

Biochemistry

journal_title

Biochemistry

authors

Semmelmann F,Hupfeld E,Heizinger L,Merkl R,Sterner R

doi

10.1021/acs.biochem.9b00286

subject

Has Abstract

pub_date

2019-06-04 00:00:00

pages

2584-2588

issue

22

eissn

0006-2960

issn

1520-4995

journal_volume

58

pub_type

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