Characterization and engineering of the adenylation domain of a NRPS-like protein: A potential biocatalyst for aldehyde generation

Meng Wang, Huimin Zhao

Research output: Contribution to journalArticlepeer-review

Abstract

The adenylation (A) domain acts as the first "gate-keeper" to ensure the activation and thioesterification of the correct monomer to nonribosomal peptide synthetases (NRPSs). Our understanding of the specificity-conferring code and our ability to engineer A domains are critical for increasing the chemical diversity of nonribosomal peptides (NRPs). We recently discovered a novel NRPS-like protein (ATEG-03630) that can activate 5-methyl orsellinic acid (5-MOA) and reduce it to 2,4-dihydroxy-5,6-dimethyl benzaldehyde. A NRPS-like protein is much smaller than multidomain NRPSs, but it still represents the thioesterification half-reaction, which is otherwise missed from a stand-alone A domain. Therefore, a NRPS-like protein may serve as a better model system for A domain engineering. Here, we characterize the substrate specificity of ATEG-03630 and conclude that the hydrogen-bond donor at the 4-position is crucial for substrate recognition. Next, we show that the substrate specificity of ATEG-03630 can be engineered toward our target substrate anthranilate via bioinformatics analysis and mutagenesis. The resultant mutant H358A increased its activity toward anthranilate by 10.9-fold, which led to a 26-fold improvement in specificity. Finally, we demonstrate one-pot chemoenzymatic synthesis of 4-hydroxybenzaldoxime from 4-hydroxybenzoic acid with high yield.

Original languageEnglish (US)
Pages (from-to)1219-1225
Number of pages7
JournalACS Catalysis
Volume4
Issue number4
DOIs
StatePublished - Apr 4 2014

Keywords

  • adenylation domain
  • aldehyde
  • NRPS-like protein
  • one-pot synthesis
  • substrate specificity engineering

ASJC Scopus subject areas

  • Catalysis

Fingerprint Dive into the research topics of 'Characterization and engineering of the adenylation domain of a NRPS-like protein: A potential biocatalyst for aldehyde generation'. Together they form a unique fingerprint.

Cite this