Discovery, activity and characterisation of an AA10 lytic polysaccharide oxygenase from the shipworm symbiont Teredinibacter turnerae

Abstract

Background The quest for novel enzymes for cellulosic biomass-degradation has recently been focussed on lytic polysaccharide monooxygenases (LPMOs/PMOs), Cu-containing proteins that catalyse the oxidative degradation of otherwise recalcitrant polysaccharides using O2 or H2O2 as a co-substrate. Results Although classical saprotrophic fungi and bacteria have been a rich source of lytic polysaccharide monooxygenases (LPMOs), we were interested to see if LPMOs from less evident bio-environments could be discovered and assessed for their cellulolytic activity in a biofuel context. In this regard, the marine shipworm Lyrodus pedicellatus represents an interesting source of new enzymes, since it must digest wood particles ingested during its natural tunnel boring behaviour and plays host to a symbiotic bacterium, Teredinibacter turnerae, the genome of which has revealed a multitude of enzymes dedicated to biomass deconstruction. Here, we show that T. turnerae encodes a cellulose-active AA10 LPMO. The 3D structure, at 1.4 Å resolution, along with its EPR spectrum is distinct from other AA10 polysaccharide monooxygenases insofar as it displays a “histidine-brace” catalytic apparatus with changes to the surrounding coordination sphere of the copper. Furthermore, TtAA10A possesses a second, surface accessible, Cu site 14 Å from the classical catalytic centre. Activity measurements show that the LPMO oxidises cellulose and thereby significantly augments the rate of degradation of cellulosic biomass by classical glycoside hydrolases. Conclusion Shipworms are wood-boring marine molluscs that can live on a diet of lignocellulose. Bacterial symbionts of shipworms provide many of the enzymes needed for wood digestion. The shipworm symbiont T. turnerae produces one of the few LPMOs yet described from the marine environment, notably adding to the capability of shipworms to digest recalcitrant polysaccharides.

The Linker Region Promotes Activity and Binding Efficiency of Modular LPMO towards Polymeric Substrate

“…Cellulose lytic monooxygenases belong to the AA9 family of CAZymes, which catalyze oxidative degradation of cellulose. LPMOs are reported to augment biomass disintegration by classical hydrolytic enzyme cocktail ( 38 ), and therefore, persistent efforts are being led to discover newer LPMOs ( 39 – 41 ). These LPMOs mainly interact with polysaccharide substrate via accessory domains, i.e., carbohydrate-binding module (CBM) ( 24 , 25 ).…”

Characterisation of the enzyme transport path between shipworms and their bacterial symbionts

“…The results are in line with the findings of O’Connor and colleagues [ 23 ], who identified numerous mannanases, mannosidases and xylanases in the caecum contents of the shipworm B. setacea . We also managed to obtain the recombinant version of a bacterial AA10 lytic polysaccharide monooxygenase (LPMO) from L. pedicellatus and characterised its ability to cleave crystalline and amorphous cellulose at the C1 position, similarly to a previously characterised bacterial AA10 from Teredinibacter turnerae [ 52 ]. Our combined omics analysis suggests that, although L. pedicellatus carries endogenous AA15 LPMO sequences, both their gene expression and protein abundance are very low in the digestive system (digestive glands, crystalline style, caecum) and that bacterial LPMOs have been co-opted towards wood digestion.…”

Characterization of a bacterial copper‐dependent lytic polysaccharide monooxygenase with an unusual second coordination sphere

“…To date, all the previously reported characterized chitin-active bacterial AA10 LPMOs display the conserved alanine residue in the second coordination sphere of the active site Cu(II) ion. The cellulose-active LPMOs CjAA10B from Cellvibrio japonicus, HcAA10A from Hahella chejuensis, and TtAA10 from Teredinibacter turnerae (all belonging to subclade IV, Figure 2a) are the only characterized bacterial AA10 LPMOs without the active site alanine, but substituted by a glycine instead [23,24,26]. Additionally, a crystal structure of a putative AA10 LPMO from Burkholderia pseudomallei with a methionine in place of the alanine was released in the Protein Data Bank (PDB accession code 3UAM).…”

“…Generally, chitin-active LPMOs regiospecifically oxidize their substrate at position C1 [6,11,15,[17][18][19]. On the contrary, some LPMOs active on cellulose were found to exclusively oxidize at position C1 or C4, while others can generate oxidation product originating from both C1 and C4 oxidation [8,16,17,[20][21][22][23][24][25][26]. As previously mentioned, LPMOs reactivity depends on a catalytically obligatory copper ion at their active site.…”