Research article summary (published 30 Jun 2009):

Characterization of endogenous and recombinant forms of laccase-2, a multicopper oxidase from the tobacco hornworm, Manduca sexta.

Full Abstract

Laccases belong to the group of multicopper oxidases that exhibit wide substrate specificity for polyphenols and aromatic amines. They are found in plants, fungi, bacteria, and insects. In insects the only known role for laccase is in cuticle sclerotization. However, extracting laccase from the insect's cuticle requires proteolysis, resulting in an enzyme that is missing its amino-terminus. To circumvent this problem, we expressed and purified full-length and amino-terminally truncated recombinant forms of laccase-2 from the tobacco hornworm, Manduca sexta. We also purified the endogenous enzyme from the pharate pupal cuticle and used peptide mass fingerprinting analysis to confirm that it is laccase-2. All three enzymes had pH optima between 5 and 5.5 when using N-acetyldopamine (NADA) or N-beta-alanyldopamine-alanyldopamine (NBAD) as substrates. The laccases exhibited typical Michaelis-Menten kinetics when NADA was used as a substrate, with K(m) values of 0.46 mM, 0.43 mM, and 0.63 mM, respectively, for the full-length recombinant, truncated recombinant, and cuticular laccases; the apparent k(cat) values were 100 min(-1), 80 min(-1), and 290 min(-1). The similarity in activity of the two recombinant laccases suggests that laccase-2 is expressed in an active form rather than as a zymogen, as had been previously proposed. This conclusion is consistent with the detection of activity in untanned pupal wing cuticle using the laccase substrate 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). Immunoblot analysis of proteins extracted from both tanned and untanned cuticle detected only a single protein of 84 kDa, consistent with the full-length enzyme. With NBAD as substrate, the full-length recombinant and cuticular laccases showed kinetics indicative of substrate inhibition, with K(m) values of 1.9 mM and 0.47 mM, respectively, and apparent k(cat) values of 200 min(-1) and 180 min(-1). These results enhance our understanding of cuticle sclerotization, and may aid in the design of insecticides targeting insect laccases.

Author information

Author/s: Dittmer, Neal T (NT); Gorman, Maureen J (MJ); Kanost, Michael R (MR);

Affiliation: Department of Biochemistry, 141 Chalmers Hall, Kansas State University, Manhattan, KS 66506-0116, USA. ndittmer(-atsign-)ksu.edu

Grants: AI070864 (Agency:NIAID NIH HHS) ; P20 RR-016464 (Agency:NCRR NIH HHS)

Journal and publication information

Publication Type: Journal Article; Research Support, N.I.H., Extramural; Research Support, U.S. Gov't, Non-P.H.S.

Journal: Insect biochemistry and molecular biology (Insect Biochem Mol Biol), published in England. (Language: eng)

Reference: 2009-Sep; vol 39 (issue 9) : pp 596-606

Dates: Created 2009/08/24; Completed 2009/10/19;

PMID: 19576986, status: MEDLINE (last retrieved date: 10/19/2009)

Sourced from the National Library of Medicine. Abstract text and other information may be subject to copyright.

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MeSH headings (categories)

This article was linked to the MeSH Headings (categories) shown below.

Amino Acid Sequence Animals Enzyme Stability Insect Proteins - chemistry; genetics; metabolism Kinetics Laccase - chemistry; genetics; metabolism

Manduca - chemistry; enzymology; genetics Molecular Sequence Data Oxidoreductases - chemistry; genetics; metabolism Recombinant Proteins - chemistry; genetics; metabolism Sequence Alignment Substrate Specificity

Note: Bold headings indicate primary MeSH headings or qualifiers.

Associated Chemicals: Insect Proteins (0) ; Recombinant Proteins (0) ; Oxidoreductases (EC 1.-) ; Laccase (EC 1.10.3.2) ; copper oxidase (EC 1.16.-)

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