Structural Basis for the ATP-dependent Configuration of Adenylation Active Site in Bacillus subtilis o-Succinylbenzoyl-CoA Synthetase

Abstract

Background: It is not clear how the adenylation active site is formed in adenylating enzymes. Results: ATP binding induces adenylate-forming conformation, creates a binding site for the carboxylate substrate, and aligns active site residues for catalysis. Conclusion: ATP configures the adenylation active site. Significance: A new structural role is revealed for ATP in forming the active adenylation conformation in the catalysis of adenylating enzymes.

Structures of the N‐Terminal Domain of PqsA in Complex with Anthraniloyl‐ and 6‐Fluoroanthraniloyl‐AMP: Substrate Activation in Pseudomonas Quinolone Signal (PQS) Biosynthesis

“…However, because the elution volume in SEC strongly depends on the shape of the protein, we also confirmed the monomeric status of PqsA NTD by small‐angle X‐ray scattering (SAXS, Figure S4). Other ANL proteins oligomerize through their N‐terminal domains (Figure S5), so these observations indicate that full‐length PqsA is most probably also a monomer in solution, as has also been concluded in previous studies …”

“…[55,61,62] Crystal packinga nalysis with PDBePISA [63] suggests that PqsA NTD is monomeric, which is in line with the results of size exclusion chromatography (SEC) of PqsA NTD .H owever,b ecause the elutionv olumei nS EC strongly depends on the shape of the protein, we also confirmed the monomeric status of PqsA NTD by small-angleX -ray scattering (SAXS, Figure S4). Other ANL proteinso ligomerize through their N-terminal domains ( Figure S5), [61,62,64,65] so these observations indicatet hat full-length PqsA is most probablya lso am onomer in solution, as has also been concluded in previouss tudies. [24]…”

Structure of the adenylation domain Thr1 involved in the biosynthesis of 4‐chlorothreonine in Streptomyces sp. OH‐5093—protein flexibility and molecular bases of substrate specificity

“…The tertiary structures arising from the varying extent of rotation of the C-domain around a pivotal position (corresponding to Asp423 in Thr1) have been assigned to the ligand-binding, adenylation, and thiolation states [19,20,[37][38][39]. Conformational transitions could be present also in the resting state, as shown in the ANL domain in o-succinylbenzoyl-CoA synthetase structure [33]. Crystallization of Thr1 in the absence of its substrates shows two independent copies in the asymmetric unit differing mainly in the relative orientation of N-and C-subdomains, monomer A and B, where the C-subdomain is rotated by 20°, in a more 'closed' conformation.…”

“…The tertiary structures arising from the varying extent of rotation of the C‐domain around a pivotal position (corresponding to Asp423 in Thr1) have been assigned to the ligand‐binding, adenylation, and thiolation states . Conformational transitions could be present also in the resting state, as shown in the ANL domain in o‐succinylbenzoyl‐CoA synthetase structure .…”

Crystal structure of the thioesterification conformation of Bacillus subtilis o-succinylbenzoyl-CoA synthetase reveals a distinct substrate-binding mode

“…Consistent with its expected use of the domain-alternation catalytic strategy, Bacillus anthracis MenE has been kinetically analyzed to adopt an ordered Bi Uni Uni Bi Ping-Pong catalytic mechanism in which the enzyme sequentially binds ATP and OSB (Bi), releases pyrophosphate (Uni) after adenylation, binds CoA (Uni) as the third substrate, and then releases OSB-CoA and AMP (Bi) after thioesterification (22). Using crystallographic methods, ligand-free MenE orthologues from various sources have been shown to adopt an open conformation with their C-domains rotated at a widely variable angle relative to the N-domains (PDB code 3ipl) (23,24), which is closed by binding of ATP through an open-closed conformational change to form the compact conformation for the first half-adenylation reaction as captured in the crystal structure of Bacillus subtilis MenE (bsMenE) in complex with ATP (24). The bsMenE-ATP structure confirms the role of transition state stabilization for a strictly conserved lysine residue (Lys-471 in bsMenE) and reveals the structural details of the carboxylate-binding site created by ATP binding.…”