Junghare, M. | Search bioRxiv

bioRxiv2023

Biochemical and structural characterisation of a family GH5 cellulase from endosymbiont of shipworm P. megotara

Cellulases play a key role in enzymatic conversion of plant cell-wall polysaccharides into simple and economically relevant sugars. The discovery of novel cellulases from exotic biological niches is of interest as they may present properties that are valuable in biorefining of lignocellulose. We have characterized a glycoside hydrolase 5 (GH5) domain of a bi-catalytic GH5-GH6 multidomain enzyme from the unusual bacterial endosymbiont Teredinibacter waterburyi of the wood-digesting shipworm Psiloteredo megotara. The cellulase enzyme, TwCel5, was produced with and without a native C-terminal family 10 carbohydrate-binding module belongs to GH5, subfamily 2. Both variants showed hydrolytic endo-activity on soluble substrates such as, {beta}-glucan, carboxymethylcellulose and konjac glucomannan. However, low activity was observed towards a crystalline form of cellulose. Interestingly, when co-incubated with a cellulose active LPMO, a clear synergy was observed that boosted hydrolysis of crystalline cellulose. The crystal structure of the GH5 catalytic domain was solved to 1.0 [A] resolution and revealed a substrate binding cleft containing a putative +3 subsite, which is uncommon in this enzyme family. The enzyme TwCel5 was active in a wide range of pH and temperatures and showed high tolerance for NaCl. This study provides an important advance on discovery new enzymes from shipworm and shed new light on biochemical and structural characterization of cellulolytic cellulase and showed boost in hydrolytic activity of cellulase on crystalline cellulose when co-incubated with cellulose active LPMO. These findings will be relevant for the development of future enzyme cocktail that may be useful for the biotechnological conversion of lignocellulose.

biochemistry↗

bioRxiv2022

Cloning, heterologous expression and characterization of o-phthalyl-CoA decarboxylase from phthalate degrading denitrifying bacterium

Phthalic acid esters (phthalates) are used as additives in various plastics and industrial applications. They are produced worldwide in huge amounts causing major pollution in the environment. Biodegradation of phthalates from the environment is an important route for their removal. In our previous work, we showed that Azoarcus sp. strain PA01 catabolizes o-phthalate via the anaerobic benzoyl-CoA pathway that involved two putative enzymes: the succinyl-CoA:o-phthalate CoA-transferase activates o-phthalate to o-phthalyl-CoA which is subsequently decarboxylated to benzoyl-CoA by o-phthalyl-CoA decarboxylase. In this work, we provide the information on the enzymes involved in the promising step of anoxic decarboxylation of o-phthalate to benzoyl-CoA. We have identified that there are two proteins are involved in decarboxylation step, of which only one does the actual decarboxylation but other one is essential. o-Phthalyl-CoA decarboxylase (PhtDa and PhtDb) encoded by the two genes PA01_00217 and PA01_00218 which catalyses the decarboxylation of activated o-phthalate to benzoyl-CoA. Both genes are originally annotated as an UbiD-like/UbiX-like protein. The gene with locus tag PA01_00217 is 1584 bp long coding for protein PhtDa (60 kDa), whereas PA01_00218 is 600 bp long codes for protein PhtDb (22 kDa). Here, we demonstrate that PhtDb is a flavin mononucleotide (FMN)-binding protein which does not function as a decarboxylase alone. Rather, PhtDb is assumed to generate a modified FMN-containing cofactor that is required by the PhtDa for decarboxylase activity. Alone, PhtDa does not function as a decarboxylase either. Recombinantly expressed PhtDa and PhtDb together showed activity for decarboxylation of o-phthalyl-CoA to benzoyl-CoA, only if PhtDb was previously incubated with FMN and dimethylallyl monophosphate. Phylogenetically, the proteins PhtDa and PhtDb are closely related to UbiD-like/UbiX-like enzymes that catalyses the decarboxylation of 4-hydroxy-3-octaprenylbenzoic acid to 2-octaprenylphenol, an intermediate step in ubiquinone biosynthesis. Furthermore, multiple sequence alignment and structural modelling of both proteins suggested that only PthDb possesses the binding site for FMN. These results strongly indicate that the flavin-containing cofactor is essential for decarboxylation of o-phthalyl-CoA to benzoyl-CoA during anaerobic o-phthalate degradation by Azoarcus sp. strain PA01.

microbiology↗