PDBsum entry 6u1h

Ligase

PDB id: 6u1h

Contents

Protein chains

324 a.a.

305 a.a.

Ligands

PO4 ×2

ADP ×4

Metals

_MG ×5

__K ×2

Waters ×311

PDB id:

6u1h

Name:

Ligase

Title:

Thermus thermophilus d-alanine-d-alanine ligase in complex with adp, phosphate, mg2+ and k+

Structure:

D-alanine--d-alanine ligase. Chain: a, b, c, d. Synonym: d-ala-d-ala ligase,d-alanylalanine synthetase. Engineered: yes

Source:

Thermus thermophilus (strain hb8 / atcc 27634 / dsm 579). Organism_taxid: 300852. Strain: hb8 / atcc 27634 / dsm 579. Gene: ddl, ttha1587. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008

Resolution:

2.20Å R-factor: 0.221 R-free: 0.265

Authors:

J.L.Pederick,J.B.Bruning

Key ref:

J.L.Pederick et al. (2020). d-Alanine-d-alanine ligase as a model for the activation of ATP-grasp enzymes by monovalent cations. J Biol Chem, 295, 7894-7904. PubMed id: 32335509 DOI: 10.1074/jbc.RA120.012936

Date:

15-Aug-19 Release date: 06-May-20

Protein chains

Q5SHZ3  (DDL_THET8) -  D-alanine--D-alanine ligase from Thermus thermophilus (strain ATCC 27634 / DSM 579 / HB8)

Seq: 319 a.a.

Struc: 324 a.a.

Protein chain

Q5SHZ3  (DDL_THET8) -  D-alanine--D-alanine ligase from Thermus thermophilus (strain ATCC 27634 / DSM 579 / HB8)

Seq: 319 a.a.

Struc: 305 a.a.

Enzyme reactions

• Enzyme class: Chains A, B, C, D: E.C.6.3.2.4 - D-alanine--D-alanine ligase.
• Pathway: Peptidoglycan Biosynthesis (Part 1)
• Reaction: 2 D-alanine + ATP = D-alanyl-D-alanine + ADP + phosphate + H⁺

2 × D-alanine + ATP = D-alanyl-D-alanine (Bound ligand (Het Group name = ADP) corresponds exactly) + ADP + phosphate + H(+) (Bound ligand (Het Group name = PO4) corresponds exactly)

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1074/jbc.RA120.012936

J Biol Chem 295:7894-7904 (2020)

PubMed id: 32335509

d-Alanine-d-alanine ligase as a model for the activation of ATP-grasp enzymes by monovalent cations.

J.L.Pederick, A.P.Thompson, S.G.Bell, J.B.Bruning.

ABSTRACT

The ATP-grasp superfamily of enzymes shares an atypical nucleotide-binding site known as the ATP-grasp fold. These enzymes are involved in many biological pathways in all domains of life. One ATP-grasp enzyme, d-alanine-d-alanine ligase (Ddl), catalyzes ATP-dependent formation of the d-alanyl-d-alanine dipeptide essential for bacterial cell wall biosynthesis and is therefore an important antibiotic drug target. Ddl is activated by the monovalent cation (MVC) K⁺, but despite its clinical relevance and decades of research, how this activation occurs has not been elucidated. We demonstrate here that activating MVCs bind adjacent to the active site of Ddl from Thermus thermophilus and used a combined biochemical and structural approach to characterize MVC activation. We found that TtDdl is a type II MVC-activated enzyme, retaining activity in the absence of MVCs. However, the efficiency of TtDdl increased ∼20-fold in the presence of activating MVCs, and it was maximally activated by K⁺ and Rb⁺ ions. A strict dependence on ionic radius of the MVC was observed, with Li⁺ and Na⁺ providing little to no TtDdl activation. To understand the mechanism of MVC activation, we solved crystal structures of TtDdl representing distinct catalytic stages in complex with K⁺, Rb⁺, or Cs⁺ Comparison of these structures with apo TtDdl revealed no evident conformational change on MVC binding. Of note, the identified MVC binding site is structurally conserved within the ATP-grasp superfamily. We propose that MVCs activate Ddl by altering the charge distribution of its active site. These findings provide insight into the catalytic mechanism of ATP-grasp enzymes.