Abstract
An increasing number of esterases is being revealed by (meta) genomic sequencing projects, but few of them are functionally/structurally characterized, especially enzymes of fungal origin. Starting from a three-member gene family of secreted putative “lipases/esterases” preferentially expressed in the symbiotic phase of the mycorrhizal fungus Tuber melanosporum (“black truffle”), we show here that these enzymes (TmelEST1-3) are dimeric, heat-resistant carboxylesterases capable of hydrolyzing various short/medium chain p-nitrophenyl esters. TmelEST2 was the most active (kcat = 2302 s−1 for p-nitrophenyl-butyrate) and thermally stable (T50 = 68.3 °C), while TmelEST3 was the only one displaying some activity on tertiary alcohol esters. X-ray diffraction analysis of TmelEST2 revealed a classical α/β hydrolase-fold structure, with a network of dimer-stabilizing intermolecular interactions typical of archaea esterases. The predicted structures of TmelEST1 and 3 are overall quite similar to that of TmelEST2 but with some important differences. Most notably, the much smaller volume of the substrate-binding pocket and the more acidic electrostatic surface profile of TmelEST1. This was also the only TmelEST capable of hydrolyzing feruloyl-esters, suggestinng a possible role in root cell-wall deconstruction during symbiosis establishment. In addition to their potential biotechnological interest, TmelESTs raise important questions regarding the evolutionary recruitment of archaea-like enzymes into mesophilic subterranean fungi such as truffles.
Highlights
An increasing number of esterases is being revealed by genomic sequencing projects, but few of them are functionally/structurally characterized, especially enzymes of fungal origin
We present here the results of a detailed functional characterization and 3D structure analysis of these three T. melanosporum enzymes, designated as TmelEST 1, 2 and 3, which are shown to be non-lypolitic Hormone Sensitive Lipases (HSL) carboxylesterases closely resembling archaeal and endosymbiotic soil bacteria esterases
As revealed by microarray analysis[21] and confirmed by RNAseq transcriptome profiling[23], the three TmelEST genes are preferentially expressed in ectomycorrhizae, with the highest relative expression levels for TmelEST3, followed by TmelEST1 and TmelEST2. The latter gene is expressed at 50% lower levels in ECM compared to TmelEST3, but is the most expressed in both free-living mycelium and fruitbodies
Summary
An increasing number of esterases is being revealed by (meta) genomic sequencing projects, but few of them are functionally/structurally characterized, especially enzymes of fungal origin. The much smaller volume of the substrate-binding pocket and the more acidic electrostatic surface profile of TmelEST1 This was the only TmelEST capable of hydrolyzing feruloyl-esters, suggestinng a possible role in root cell-wall deconstruction during symbiosis establishment. All HSL esterases (EST) identified so far, display a characteristic α/β hydrolase fold[12], with a central β-sheet surrounded by multiple α-helices This serves as a stable core bearing most of the amino acid substitutions, loop insertions and deletions that have contributed to catalytic function diversification during evolution. Three distinct genes automatically annotated as “secreted lipases/esterases”[21] are preferentially expressed in the symbiotic phase of Tuber melanosporum ( known as “black truffle”), a mycorrhizal ascomycete whose genome and methylome have been sequenced[21,22] This fungus, which forms mutualistic interactions with various host-plants, is characterized by a strictly subterranean habitat and three distinct lifecycle stages: free-living mycelium (FLM), fruiting bodies (FB; known as “truffles”) and the ectomycorrhizal (ECM) symbiotic stage
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