PDBsum entry 6ecc

Hydrolase

PDB id: 6ecc

Contents

Protein chain

231 a.a.

Waters ×171

PDB id:

6ecc

Name:

Hydrolase

Title:

Vlm2 thioesterase domain wild type structure 2

Structure:

Vlm2. Chain: a. Fragment: thioesterase domain (unp residues 2368-2655). Engineered: yes

Source:

Streptomyces tsusimaensis. Organism_taxid: 285482. Atcc: 15141. Gene: vlm2. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008

Resolution:

1.80Å R-factor: 0.177 R-free: 0.190

Authors:

D.A.Alonzo,T.M.Schmeing

Key ref:

N.Huguenin-Dezot et al. (2019). Trapping biosynthetic acyl-enzyme intermediates with encoded 2,3-diaminopropionic acid. Nature, 565, 112-117. PubMed id: 30542153

Date:

07-Aug-18 Release date: 12-Dec-18

Protein chain

Q1PSF3  (Q1PSF3_9ACTN) -  Vlm2 from Streptomyces tsusimaensis

Seq: 2655 a.a.

Struc: 231 a.a.

Enzyme reactions

• Enzyme class: E.C.3.1.2.- - ?????

Nature 565:112-117 (2019)

PubMed id: 30542153

Trapping biosynthetic acyl-enzyme intermediates with encoded 2,3-diaminopropionic acid.

N.Huguenin-Dezot, D.A.Alonzo, G.W.Heberlig, M.Mahesh, D.P.Nguyen, M.H.Dornan, C.N.Boddy, T.M.Schmeing, J.W.Chin.

ABSTRACT

Many enzymes catalyse reactions that proceed through covalent acyl-enzyme (ester or thioester) intermediates¹. These enzymes include serine hydrolases^2,3 (encoded by one per cent of human genes, and including serine proteases and thioesterases), cysteine proteases (including caspases), and many components of the ubiquitination machinery^4,5. Their important acyl-enzyme intermediates are unstable, commonly having half-lives of minutes to hours⁶. In some cases, acyl-enzyme complexes can be stabilized using substrate analogues or active-site mutations but, although these approaches can provide valuable insight^7-10, they often result in complexes that are substantially non-native. Here we develop a strategy for incorporating 2,3-diaminopropionic acid (DAP) into recombinant proteins, via expansion of the genetic code¹¹. We show that replacing catalytic cysteine or serine residues of enzymes with DAP permits their first-step reaction with native substrates, allowing the efficient capture of acyl-enzyme complexes that are linked through a stable amide bond. For one of these enzymes, the thioesterase domain of valinomycin synthetase¹², we elucidate the biosynthetic pathway by which it progressively oligomerizes tetradepsipeptidyl substrates to a dodecadepsipeptidyl intermediate, which it then cyclizes to produce valinomycin. By trapping the first and last acyl-thioesterase intermediates in the catalytic cycle as DAP conjugates, we provide structural insight into how conformational changes in thioesterase domains of such nonribosomal peptide synthetases control the oligomerization and cyclization of linear substrates. The encoding of DAP will facilitate the characterization of diverse acyl-enzyme complexes, and may be extended to capturing the native substrates of transiently acylated proteins of unknown function.