Harzianic Acid from Trichoderma afroharzianum Is a Natural Product Inhibitor of Acetohydroxyacid Synthase

Abstract

Acetohydroxyacid synthase (AHAS) is the first enzyme in the branched-chain amino acid biosynthetic pathway and is a validated target for herbicide and fungicide development. Here we report harzianic acid (HA, 1) produced by the biocontrol fungus Trichoderma afroharzianum t-22 (Tht22) as a natural product inhibitor of AHAS. The biosynthetic pathway of HA was elucidated with heterologous reconstitution. Guided by a putative self-resistance enzyme in the genome, HA was biochemically demonstrated to be a selective inhibitor of fungal AHAS, including those from phytopathogenic fungi. In addition, HA can inhibit a common resistant variant of AHAS in which the active site proline is mutated. Structural analysis of AHAS complexed with HA revealed the molecular basis of competitive inhibition, which differs from all known commercial AHAS inhibitors. The alternative binding mode also rationalizes the selectivity of HA, as well as effectiveness toward resistant mutants. A proposed role of HA biosynthesis by Tht22 in the rhizosphere is discussed based on the data.

New Approaches to Manage Asian Soybean Rust (Phakopsora pachyrhizi) Using Trichoderma spp. or Their Antifungal Secondary Metabolites

“…Literature review revealed that the substance corresponding to this molecular formula might be harzianic acid (HA) [42,[47][48][49][50]. In addition, the UV absorption of peak 1 with λ max 238 and 358 nm (Figure S1) was similar to those found for HA in multiple reports [42,[50][51][52]. The identity of HA was confirmed using LC/MS/MS spectra in the positive ionization mode which showed distinct product ions (Figure 1c 1 ,c 2 ) corresponding to the already predicted mass spectra of HA [47,53].…”

Structural basis of resistance to herbicides that target acetohydroxyacid synthase

“…It is therefore suggested that future design efforts should be concentrated on inhibitors that avoid entering that space. In this regard, it is worth pointing out the mode of binding of the recently isolated natural product inhibitor of AHAS, harzianic acid 28 . This compound not only takes a conformation avoiding the region where the oxidative side-reaction occurs, but also makes interactions within a region of the catalytic pocket that has not been used by any of the existing commercial herbicides.…”

ThpacC Acts as a Positive Regulator of Homodimericin A Biosynthesis and Antifungal Activities of Trichoderma harzianum 3.9236

“…harzianum. , The biosynthetic pathway of HA was newly elucidated in T. afroharzianum, and HA was biochemically characterized to be a selective inhibitor of fungal acetohydroxyacid synthase (AHAS), which was a validated target for fungicide development . There is no obvious difference between the expression levels of WT and KO- ThpacC concerning the biosynthetic core genes for HA production, which supported that HA production could be ThpacC-independent and pH-dependent.…”