Mapping the Allosteric Communication Network of Aminodeoxychorismate Synthase

Semmelmann, Florian; Straub, Kristina; Nazet, Julian; Rajendran, Chitra; Merkl, Rainer; Sterner, Reinhard

doi:10.1016/j.jmb.2019.05.021

Cited by 13 publications

(19 citation statements)

References 53 publications

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“…This concurs with the need of the nucleophile for catalysis. 131 The allosteric activation of the amidotransferase domain ensures that glutamine is only consumed if chorismate is available for conversion into the corresponding amino derivative ADC. Trp abolishes catalysis in anthranilate synthases allosterically by effecting an ammonia channel to the active site without major structural changes.…”

Section: The Metabolic Flux Into the Different Branches Starting From (Iso)chorismate Is Directed By Allosteric Regulationmentioning

confidence: 99%

Chorismate- and isochorismate converting enzymes: versatile catalysts acting on an important metabolic node

2021

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Section: The Metabolic Flux Into the Different Branches Starting From (Iso)chorismate Is Directed By Allosteric Regulationmentioning

confidence: 99%

Chorismate- and isochorismate converting enzymes: versatile catalysts acting on an important metabolic node

2021

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“…This missense mutation resulted in a K546E exchange in the PabB domain within the highly conserved ADC synthase component I motif PIKGT. Lysine in the wildtype (wt) covalently binds to the C 2 of chorismate to initiate with the ammonia-group of glutamine the enzymatic formation of ADC ( [28][29][30][31], Fig. 1a).…”

Section: Genes Pab1 and Ade8 In C Cinereamentioning

confidence: 99%

“…Other residues affecting enzymatic activities and bonding to PabB are marked with grey squares. The position of a stabilizing residue stretch called oxyanion hole is underlaid in light blue, a sequence stretch for chorismate signal transfer in olive [29,30,75]. Red letters in PabB mark helical regions, blue letters β-sheets.…”

Section: Figmentioning

confidence: 99%

“…Symbols * in red and black mark (predicted) active site residues and Mg 2+ -binding residues in two chorismate-interacting helices, respectively. Triangles in black indicate residues that contact the bound tryptophan and grey squares further residues where mutations affect functionality [28][29][30][31]76]. b Red, blue, green and magenta letters mark the N, B, A, and C domains of PurD.…”

Section: Figmentioning

confidence: 99%

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Selection markers for transformation of the sequenced reference monokaryon Okayama 7/#130 and homokaryon AmutBmut of Coprinopsis cinerea

Dörnte

Peng

Fang

et al. 2020

Fungal Biol Biotechnol

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Background Two reference strains have been sequenced from the mushroom Coprinopsis cinerea, monokaryon Okayama 7/#130 (OK130) and the self-compatible homokaryon AmutBmut. An adenine-auxotrophy in OK130 (ade8-1) and a para-aminobenzoic acid (PABA)-auxotrophy in AmutBmut (pab1-1) offer selection markers for transformations. Of these two strains, homokaryon AmutBmut had been transformed before to PABA-prototrophy and with the bacterial hygromycin resistance marker hph, respectively. Results Gene ade8 encodes a bifunctional enzyme with an N-terminal glycinamide ribonucleotide synthase (GARS) and a C-terminal aminoimidazole ribonucleotide synthase (AIRS) domain required for steps 2 and 5 in the de novo biosynthesis of purines, respectively. In OK130, a missense mutation in ade8-1 rendered residue N231 for ribose recognition by the A loop of the GARS domain into D231. The new ade8+ vector pCcAde8 complements the auxotrophy of OK130 in transformations. Transformation rates with pCcAde8 in single-vector and co-transformations with ade8+-selection were similarly high, unlike for trp1+ plasmids which exhibit suicidal feedback-effects in single-vector transformations with complementation of tryptophan synthase defects. As various other plasmids, unselected pCcAde8 helped in co-transformations of trp1 strains with a trp1+-selection vector to overcome suicidal effects by transferred trp1+. Co-transformation rates of pCcAde8 in OK130 under adenine selection with nuclear integration of unselected DNA were as high as 80% of clones. Co-transformation rates of expressed genes reached 26–42% for various laccase genes and up to 67% with lcc9 silencing vectors. The bacterial gene hph can also be used as another, albeit less efficient, selection marker for OK130 transformants, but with similarly high co-transformation rates. We further show that the pab1-1 defect in AmutBmut is due to a missense mutation which changed the conserved PIKGT motif for chorismate binding in the C-terminal PabB domain to PIEGT in the mutated 4-amino-4-deoxychorismate synthase. Conclusions ade8-1 and pab1-1 auxotrophic defects in C. cinerea reference strains OK130 and AmutBmut for complementation in transformation are described. pCcAde8 is a new transformation vector useful for selection in single and co-transformations of the sequenced monokaryon OK130 which was transformed for the first time. The bacterial gene hph can also be used as an additional selection marker in OK130, making in combination with ade8+ successive rounds of transformation possible.

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“…We recently analyzed the allosteric communication pathways within 4-amino-4-deoxychorsimate synthase (ADCS), 15 a member of class I GATases. We identified four conserved allosteric signaling pathways that lead to activity-inducing conformational changes at the glutaminase subunit PabA as a consequence of the binding of the substrate chorismate to the synthase subunit PabB.…”

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confidence: 99%

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

et al. 2019

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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.

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Mapping the Allosteric Communication Network of Aminodeoxychorismate Synthase

Cited by 13 publications

References 53 publications

Chorismate- and isochorismate converting enzymes: versatile catalysts acting on an important metabolic node

Chorismate- and isochorismate converting enzymes: versatile catalysts acting on an important metabolic node

Selection markers for transformation of the sequenced reference monokaryon Okayama 7/#130 and homokaryon AmutBmut of Coprinopsis cinerea

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

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