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. 2002 Sep 17;99(19):12120-5.
doi: 10.1073/pnas.182156699. Epub 2002 Sep 9.

Crystal structure of DhbE, an archetype for aryl acid activating domains of modular nonribosomal peptide synthetases

Jurgen J May  1 , Nadine Kessler, Mohamed A Marahiel, Milton T Stubbs
Affiliations

Crystal structure of DhbE, an archetype for aryl acid activating domains of modular nonribosomal peptide synthetases

Jurgen J May et al. Proc Natl Acad Sci U S A. .

Abstract

The synthesis of the catecholic siderophore bacillibactin is accomplished by the nonribosomal peptide synthetase (NRPS) encoded by the dhb operon. DhbE is responsible for the initial step in bacillibactin synthesis, the activation of the aryl acid 2,3-dihydroxybenzoate (DHB). The stand-alone adenylation (A) domain DhbE, the structure of which is presented here, exhibits greatest homology to other NRPS A-domains, acyl-CoA ligases and luciferases. It's structure is solved in three different states, without the ligands ATP and DHB (native state), with the product DHB-AMP (adenylate state) and with the hydrolyzed product AMP and DHB (hydrolyzed state). The 59.9-kDa protein folds into two domains, with the active site at the interface between them. In contrast to previous proposals of a major reorientation of the large and small domains on substrate binding, we observe only local structural rearrangements. The structure of the phosphate binding loop could be determined, a motif common to many adenylate-forming enzymes, as well as with bound DHB-adenylate and the hydrolyzed product DHB*AMP. Based on the structure and amino acid sequence alignments, an adapted specificity conferring code for aryl acid activating domains is proposed, allowing assignment of substrate specificity to gene products of previously unknown function.

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Figures

Fig 1.
Fig 1.
(A) Bacillibactin NRPS cluster from B. subtilis with the corresponding domain organization of synthetase modules. ICL, isochorismatase; C, condensation domain; T, thiolation domain. (B) Structure of the trilactone Bacillibactin, with one of the catecholic moiety activated by DhbE shaded in gray. (C) The DhbE-dependent aryl acid adenylation in peptide synthesis is an ATP-consuming process leading to a protein-bound adenylate.
Fig 2.
Fig 2.
(A) Stereo presentation of the DhbE structure (adenylated state) in presence of the product DHB-adenylate (CPK model). The core motifs are shown in blue, the N-terminal domain is shown in green, and the lid domain is shown in red. The N-terminal domain can be further divided into three, defined by a 6-strand/5-helix subdomain (a), an 8-strand/6-helix part (b), and a 5-stranded β-barrel (c). The N-terminal helix nestles between a and c. Core motif locations are also indicated. (B) Focus on the substrate-binding pocket in the adenylated state. The 10 residues determining substrate specificity are labeled in blue, those coordinating AMP in black. The side chains of these residues and the product are shown as ball-and-stick model.
Fig 3.
Fig 3.
(A) Close-up of the catalytic site of DhbE and PheA in the region between core A4 and core A5 (see B). The route of the main chain is represented as solid ribbon; the ligands Phe (orange), DHB (red), and AMP are represented as sticks. Residues that influence the main chain route or are involved in substrate binding are labeled. (B) Secondary structure and sequence comparison of DhbE, PheA, and Luciferase. The core motifs of NRPS A domains are boxed and labeled. The asterisks in the primary sequence indicate the 10 residues responsible for substrate specificity of the A domains.
Fig 4.
Fig 4.
Experimental (2Fo − Fc) electron density contoured at 0.9 σ, showing the active site region of DhbE. Phases were calculated after a cycle of simulated annealing omitting residues shown; remaining electron density is not shown for the sake of clarity. (A) Electron density of the p-loop in the native state (residues Ser-190–Lys-198); the loop is located above the active site, with a break in the density at Gly-191. (B) In the presence of DHB and AMP (orange, hydrolyzed state), the loop moves away from the position shown in A. Furthermore, the side chain of Arg-428 extends down to interact with the α-phosphate group. (C) On adenylate formation (orange, adenylated state), the loop returns to a conformation similar to that seen in A, as does the side chain of Arg-428, where it is in contact with Asp-413.
Fig 5.
Fig 5.
Determination of the specificity conferring code of ca-activating domains. The primary sequence between core A4 and A5 (see Fig. 3) of nine ca activating domains are aligned by using the Clustal method. Based on the structural data of DhbE, extraction of the 10 residues conferring the substrate specificity leads to the identification of the signature sequence of ca activating A domain. The asterisks define the residues that allow discrimination between DHB and SAL.
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