Phenol-oxidizing laccases from the termite gut.
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 - mass spectrometry) analysis of RfLacA identified bound metals consisting mainly of copper (∼4 copper molecules per laccase protein molecule and ∼3 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.
Coy
M R
MR
Department of Entomology and Nematology, University of Florida, Gainesville, FL 32611, USA. birdpodz@yahoo.com
Salem
T Z
TZ
Denton
J S
JS
Kovaleva
E S
ES
Liu
Z
Z
Barber
D S
DS
Campbell
J H
JH
Davis
D C
DC
Buchman
G W
GW
Boucias
D G
DG
Scharf
M E
ME
D000818:Animals; D019476:Insect Proteins / Q000235:genetics / Q000378:metabolism*; D007422:Intestines / Q000201:enzymology; D020049:Isoptera / Q000145:classification / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D042845:Laccase / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D019800:Phenol / Q000378:metabolism*; D010802:Phylogeny
D019476:Insect Proteins; D019800:Phenol; D042845:Laccase