ARS | Publication request: Phenol-oxidizing laccases from the termite gut

Submitted to: Insect Biochemistry and Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 21, 2010
Publication Date: August 5, 2010
Citation: Coy, M.R., Salem, T.Z., Denton, J.S., Kovaleva, E.S., Liu, Z., Barber, D.S., Campbell, J.H., Davis, D.C., Buchman, G.W., Boucias, D.G., Scharf, M.E. 2010. Phenol-oxidizing laccases from the termite gut. Insect Biochemistry and Molecular Biology. 40(10):723-732.

Interpretive Summary: Termites consume wood, which is an extraordinarily complex mixture of molecules, as their primary source of nutrition. Most organisms do not possess the enzymes necessary to break down wood, but termites possess a battery of enzymes which facilitate its breakdown simple sugars, which are then used as an energy source as typical in other organisms. One such group of enzymes, the laccases, which was originally discovered in wood-degrading fungi, was discovered in the eastern subterranean termite, Reticulitermes flavipes. Previously, laccases were identified in insects and were implicated in the hardening process of the insect exoskeleton. However, in this case laccase was found to be expressed in the midgut of the termite, in a region where digestion takes place, suggesting a potential role in the breakdown of wood. This work characterized the genes expressing laccases in R. flavipes, determined the spatial expression of these genes, determined the evolutionary relationship of these laccases to other laccases from diverse organisms, and finally characterized the enzymatic properties of the laccases.

Technical Abstract: cDNAs encoding two gut laccase isoforms (RfLacA and RfLacB) were sequenced from the termite Reticulitermes flavipes. Phylogenetic analyses comparing translated R. flavipes laccases to 67 others from prokaryotes and eukaryotes indicate that the R. flavipes laccases are evolutionarily unique. Alignments with crystallography-verified laccases confirmed that peptide motifs involved in metal binding are 100% conserved in both isoforms. Laccase transcripts and phenoloxidase activity were most abundant in symbiont-free salivary gland and foregut tissue, verifying that the genes and activities are host-derived. Using a baculovirus-insect expression system, the two isoforms were functionally expressed with histidine tags and purified to near homogeneity. ICP-MS (inductively coupled plasma e mass spectrometry) analysis of RfLacA identified bound metals consisting mainly of copper (w4 copper molecules per laccase protein molecule and w3 per histidine tag) with lesser amounts of calcium, manganese and zinc. Both recombinant enzyme preparations showed strong activity towards the lignin monomer sinapinic acid and four other phenolic substrates. By contrast, both isoforms displayed much lower or no activity against four melanin precursors, suggesting that neither isoform is involved in integument formation. Modification of lignin alkali by the recombinant RfLacA preparation was also observed. These findings provide evidence that R. flavipes gut laccases are evolutionarily distinct, host-derived, produced in the salivary gland, secreted into the foregut, bind copper, and play a role in lignocellulose digestion. These findings contribute to a better understanding of termite digestion and gut physiology, and will assist future translational studies that examine the contributions of individual termite enzymes in lignocellulose digestion.