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Determination of the MurD mechanism through crystallographic analysis of enzyme complexes1

https://doi.org/10.1006/jmbi.1999.2800Get rights and content

Abstract

UDP-N-acetylmuramoyl-l-alanine:d-glutamate (MurD) ligase catalyses the addition of d-glutamate to the nucleotide precursor UDP-N-acetylmuramoyl-l-alanine (UMA). The crystal structures of three complexes of Escherichia coli MurD with a variety of substrates and products have been determined to high resolution. These include (1) the quaternary complex of MurD, the substrate UMA, the product ADP, and Mg2+, (2) the quaternary complex of MurD, the substrate UMA, the product ADP, and Mn2+, and (3) the binary complex of MurD with the product UDP-N-acetylmuramoyl-l-alanine-d-glutamate (UMAG). The reaction mechanism supported by these structures proceeds by the phosphorylation of the C-terminal carboxylate group of UMA by the γ-phosphate group of ATP to form an acyl-phosphate intermediate, followed by the nucleophilic attack by the amino group of d-glutamate to produce UMAG. A key feature in the reaction intermediate is the presence of two magnesium ions bridging negatively charged groups.

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Cited by (137)

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    These divalent cations are not required for the binding of the substrates UMA and ADP, but play a crucial role in the ligase action of the MurD enzyme (Kotnik et al., 2007). The principle residues of the MurD enzyme of E. coli which take part while interacting with the substrate UMA involves amino acid residues Leu15, Thr16, Asp35, Thr36, Arg37, Gly73, and Asn138, while His183 interacts with Mg2+(Bertrand et al., 1997, 1999, 2000; Bertrand et al., 1999). At the same time, ADP interacts with Gly114, Lys115, Ser116, Thr117, Asn271, Arg302, and Asp317 residues.

  • Conformational dynamics of a multidomain protein by neutron scattering and computational analysis

    2021, Biophysical Journal
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    A MurD has 437 amino acid residues and consists of three domains, which are defined as from 1 to 93 (D1), 94–298 (D2), and 299–437 (D3) in the amino acid sequence (29). So far, numerous crystal structures of MurD have been identified (29–31), including the apo state (Protein Data Bank (PDB): 1E0E (29)), intermediate states (PDB: 5A5E and 5A5F (32)), and compound 1 (N-({3-[({4-[(Z)-(2,4-dioxo-1,3-thiazolidin-5-ylidene)methyl]phenyl}amino)methyl]phenyl}carbonyl)-D-glutamic acid) inhibitor-bound state (PDB: 2X5O (33)). These crystal structures are shown in Fig. 1 and suggest that one of the three domains of MurD, i.e., domain 3 (D3), undergoes drastic conformational change from the open form to the closed one via the semiclosed one upon ligand bindings, which is relevant to the enzymatic function (12,28–33).

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Edited by R. Huber

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