Structure ofD-alanine-D-alanine ligase fromYersinia pestis: nucleotide phosphate recognition by the serine loop

Abstract

D-Alanyl-D-alanine is an essential precursor of bacterial peptidoglycan and is synthesized by D-alanine-D-alanine ligase (DDL) with hydrolysis of ATP; this reaction makes DDL an important drug target for the development of antibacterial agents. Five crystal structures of DDL from Yersinia pestis (YpDDL) were determined at 1.7-2.5 Å resolution: apo, AMP-bound, ADP-bound, adenosine 5'-(β,γ-imido)triphosphate-bound, and D-alanyl-D-alanine- and ADP-bound structures. YpDDL consists of three domains, in which four loops, loop 1, loop 2 (the serine loop), loop 3 (the ω-loop) and loop 4, constitute the binding sites for two D-alanine molecules and one ATP molecule. Some of them, especially the serine loop and the ω-loop, show flexible conformations, and the serine loop is mainly responsible for the conformational change in substrate nucleotide phosphates. Enzyme-kinetics assays were carried out for both the D-alanine and ATP substrates and a substrate-binding mechanism was proposed for YpDDL involving conformational changes of the loops.

Insights into the Inhibition of Aeromonas hydrophila d-Alanine–d-Alanine Ligase by Integration of Kinetics and Structural Analysis

“…43 Sequence Analysis. The sequence of AhDdl was aligned with D-alanine−D-alanine ligase sequences from Thermus caldophilus (TcDdl, 33.90%), 44 Staphylococcus aureus COL (SaDdl, 32.29%), 20 Thermus thermophilus HB8 (TtDdl, 33.91%), 45 Yersinia pestis (YpDdl, 32.21%), 46 and Mycobacterium tuberculosis (MtDdl, 36.22%) 15 (Figure 4). The identity ranged from 32.21% identity for YpDdl to 36.22% identity for MtDdl; nevertheless, the structure-based sequence alignments of AhDdl, TcDdl, SaDdl, TtDdl, YpDdl, and MtDdl showed that the active sites were conserved in the Ddls (Figure 4) and the lengths of loop 1 (Gly13-His17), loop 2 (Ala162-Ser167), and loop 4 (Phe297-Met305) were the same.…”

“…The asymmetric unit with P2 1 2 1 2 1 symmetry contained two subunits arranged as a face-to-face homodimer, which was mainly maintained by three helices (α2, α3, and α4). Like other Ddls (TcDdl, PDB ID: 2FB9; 44 SaDdl, PDB ID: 2I80; 20 TtDdl, PDB ID: 2ZDG, 2ZDH, 2ZDQ; 45 YpDdl, PDB ID: 4ZQI 46 and MtDdl, PDB ID: 3LWB 15 ), each subunit of AhDdl was composed of three distinct domains, an Nterminal domain (domain 1, Met1-Asp106), a central domain (domain 2, Ala107-Gly200), and a C-terminal domain (domain 3, Pro201-Val329) (Figure 5A,B). Data collection and refinement statistics are summarized in Table 1.…”

Catalytic Intermediate Crystal Structures of Cysteine Desulfurase from the Archaeon Thermococcus onnurineus NA1

“…The open reading frame sequence encoding ToCDS protein from Thermococcus onnurineus NA1 was amplified by PCR using the genomic DNA isolated from Thermococcus onnurineus NA1 as a template according to a previously described method [19]. The sequences of the oligonucleotide primers were designed based on the data on genome sequences of Thermococcus onnurineus NA1 from the NCBI website.…”

Discovery of novel antibacterial agents: Recent developments in D‐alanyl‐D‐alanine ligase inhibitors

“…Ddl involved in peptidoglycan precursor biosynthesis is expressed by the ddl gene and is widely distributed in Gram‐positive and Gram‐negative bacteria (Albar et al., 1992; Tran et al., 2016). It has two isoenzymes, D‐alanine‐D‐alanine ligase A (DdlA) and D‐alanine‐D‐alanine ligase B (DdlB; Batson et al., 2010), which display similar kinetic characteristics, substrate specificity, and sensitivity to known Ddl inhibitors (Daub et al., 1988; Fan et al., 1997; Zawadzke et al., 1991).…”