Discovery of a Novel Glucuronan Lyase System in Trichoderma parareesei

Abstract

Glucuronan lyases (EC 4.2.2.14) catalyze depolymerization of linear β-(1, 4)-polyglucuronic acid (glucuronan). Only a few glucuronan lyases have been characterized until now, most of them originating from bacteria. Here we report the discovery, recombinant production, and functional characterization of the full complement of six glucuronan specific polysaccharide lyases in the necrotic mycoparasite Trichoderma parareesei . The enzymes belong to four different polysaccharide lyase families and have different reaction optima and glucuronan degradation profiles. Four of them showed endo-lytic action and two, TpPL8A and TpPL38A, displayed exo-lytic action. NMR revealed that the monomeric end-product from TpPL8A and TpPL38A underwent spontaneous rearrangements to tautomeric forms. Proteomic analysis of the secretomes from T. parareesei growing on pure glucuronan and lyophilized A. bisporus fruiting bodies, respectively, showed secretion of five of the glucuronan lyases and HPAEC-PAD analysis confirmed the presence of glucuronic acid in the A. bisporus fruiting bodies. By systematic genome annotation of more than 100 fungal genomes and subsequent phylogenetic analysis of the putative glucuronan lyases, we show that glucuronan lyases occur in several ecological and taxonomic groups in the fungal kingdom. Our findings suggest that a diverse repertoire of glucuronan lyases is a common trait among Hypocreales spp. with mycoparasitic and entomopathogenic lifestyles. IMPORTANCE This paper reports the discovery of a set of six complementary glucuronan lyase enzymes in the mycoparasite Tricoderma parareseei . Apart from the novelty of the discovery of these enzymes in T. parareesei, the key importance of the study is the finding that the majority of these lyases are induced when T. parareseei is inoculated on Basidiomycete cell walls that contain glucuronan. The study also reveals putative glucuronan lyase encoding genes in a wealth of other fungi that furthermore points at fungal cell wall glucuronan being a target C-source for many types of fungi. In a technical context, the findings may lead to controlled production of glucuronan oligomers for advanced pharmaceutical applications and pave the way for development of new fungal biocontrol agents.

Glucuronan lyases from family PL7 use a Tyr/Tyr syn β-elimination catalytic mechanism for glucuronan breakdown

“…TpPL7B appears to prefer to make dimers suggesting a processive mode of action, which is often observed for CAZymes with a tunnel shaped active site, 13 however, TpPL7B is an endolytic enzyme. 1 All in all, this suggests that the loops constituting the active site tunnel of TpPL7B closes upon substrate binding as suggested for other PL7 members, [10][11][12] but remains closed during several catalytic cycles. Processive enzymes are often slow due to the time it takes to translocate the substrate along a single axis, 14 which is in agreement with TpPL7B's 131-times lower k cat compared to TpPL7A (Table S2, ESI †).…”

“…The six GLs belong to four different CAZy families (PL7, PL8, PL20 and PL38) both exo-and endo-acting, which constitutes all together an efficient machinery for the complete degradation of b-D-1,4-glucuronan. 1 Polysaccharide lyases (PLs) are presently classified into 42 families in the Carbohydrate-Active enZymes database (CAZy) (https://www.cazy.org) 2 and to date, GLs have been identified in families PL5, PL7, PL8, PL14, PL20, PL31 and PL38. In general, PLs act using a lytic b-elimination mechanism generating a 4, 5-unsaturated hexuronic acid product (denoted D) and a new reducing end at the cleavage site.…”

“…The discrete differences observed for poly-uronic acid structures result in the requirement of different enzymes for their respective depolymerization. Within the PL7 family there are three characterized members which are specific towards b-D-1, 4-glucuronan 1 and the remaining 57 characterized members act on alginate. 2 The suggested mechanism for most PL7 alginate lyases is an anti b-elimination mechanism involving a histidine acting as the base and a tyrosine acting as the acid 8 (Fig.…”

Polyphenol Oxidase Products Are Priming Agents for LPMO Peroxygenase Activity

“…The cloning and expression methodology was based on Pilgaard et al. (2022), [39] with modifications. Genes encoding Nc AA9C (UniProt Q7SHI8) and Mt PPO7 (UniProt A0A1C9CXH9) were chemically synthesized after codon‐optimization for expression in Pichia pastoris (GenScript, Piscataway, NJ, USA).…”

Genome Analysis of a Novel Polysaccharide-Degrading Bacterium Paenibacillus algicola and Determination of Alginate Lyases

“…In the CAZy database, a total of 1383 proteins are classified as PL38 family members. Only two of them, CUL-I and TpPL38A, have been characterized as endoβ-1,4-glucuronan lyase, and CUL-I also exhibits alginate lyase activity as well [32,33]. Detailed analysis of the CAZyme information of Paenibacillus species was rarely reported.…”