Abstract
3-deoxy-D-arabino-heptulosonate-7-phosphate-synthase (DAHPS) is the first enzyme of the shikimate pathway and is responsible for the synthesis of aromatic amino acids in microorganisms. This pathway is an attractive target for antimicrobial drugs. In Bacillus subtilis, the N-terminal domain of the bifunctional DAHPS enzyme belongs to an AroQ class of chorismate mutase and is functionally homologous to the downstream AroH class chorismate mutase. This is the first structure of chorismate mutase, AroQ (BsCM_2) enzyme from Bacillus subtilis in complex with citrate and chlorogenic acid at 1.9 Å and 1.8 Å resolution, respectively. This work provides the structural basis of ligand binding into the active site of AroQ class of chorismate mutase, while accompanied by the conformational flexibility of active site loop. Molecular dynamics results showed that helix H2′ undergoes uncoiling at the first turn and increases the mobility of loop L1′. The side chains of Arg45, Phe46, Arg52 and Lys76 undergo conformational changes, which may play an important role in DAHPS regulation by the formation of the domain-domain interface. Additionally, binding studies showed that the chlorogenic acid binds to BsCM_2 with a higher affinity than chorismate. These biochemical and structural findings could lead to the development of novel antimicrobial drugs.
Highlights
Chorismic acid, the precursor for a wide range of aromatic compounds, including aromatic amino acids, folate, and ubiquinones, is essential for the survival of bacteria and is synthesized through the shikimate pathway[1]
In B. subtilis, regulation of DAHP synthase is thought to be controlled by its N-terminal domain (BsCM_2), which has residual chorismate mutase activity[26, 27, 29]
We have reported the crystal structures of the BsCM_2 domain of DAHP synthase from B. subtilis in complex with citrate and chlorogenic acid. These crystal structures provide crucial information regarding the interaction of ligands with the protein and subsequent conformational changes, which may be responsible for allosteric regulation of DAHP synthase activity
Summary
The precursor for a wide range of aromatic compounds, including aromatic amino acids, folate, and ubiquinones, is essential for the survival of bacteria and is synthesized through the shikimate pathway[1]. A DAHPS enzyme from Listeria monocytogenes has an N-terminal CM, type II domain (LmCM), which regulates the enzyme’s activity through binding of chorismate or prephenate[9]. The full-length protein exhibits the activity of both enzymes, but interestingly, in the absence of the CM domain, the DAHPS activity is significantly increased, whereas the separated CM domain is catalytically less active[25] This observation clearly implies that the CM domains interacts with the DAHPS domain and possess a regulatory role in the wild-type enzyme. Based on the prephenate bound DAHPS crystal structure, in addition to SAXS analysis, Nazmi et al proposed that in the unbound state, the CM domain adopts a open conformation and allows the DAHPS substrate to access its active site. This structural rearrangement results in obstruction of the DAHPS active site thereby inhibits its activity
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