title: Cloning and expressing a highly functional and substrate specific farnesoic acid o-methyltransferase from the Asian citrus psyllid (Diaphorina citri Kuwayama).
abstract: The Asian citrus psyllid, Diaphorina citri, transmits a phloem-limited bacterium, Candidatus 'Liberibacter' asiaticus that causes citrus greening disease. Because juvenile hormone (JH) plays an important role in adult and nymphal development, we studied the final steps in JH biosynthesis in D. citri. A putative JH acid methyltransferase ortholog gene (jmtD) and its cognate cDNA were identified by searching D. citri genome database. Expression analysis shows expression in all life stages. In adults, it is expressed in the head-thorax, (containing the corpora allata), and the abdomen (containing ovaries and male accessory glands). A 3D protein model identified the catalytic groove with catalytically active amino acids and the S-adenosyl methionine (SAM)-binding loop. The cDNA was expressed in Escherichia coli cells and the purified enzyme showed high preference for farnesoic acid (FA) and homoFA (kcat of 0.752 × 10(-3) and 0.217 × 10(-3) s(-1), respectively) as compared to JH acid I (JHA I) (cis/trans/cis; 2Z, 6E, 10cis), JHA III (2E, 6E, 10cis), and JHA I (trans/cis/cis; 2E, 2Z, 10cis) (kcat of 0.081 × 10(-3), 0.013 × 10(-3), and 0.003 × 10(-3) s(-1), respectively). This suggests that this ortholog is a DcFA-o-methyl transferase gene (fmtD), not a jmtD, and that JH biosynthesis in D. citri proceeds from FA to JH III through methyl farnesoate (MF). DcFA-o-MT does not require Ca(2+), Mg(2+) or Zn(2+), however, Zn(2+) (1 mM) completely inhibits the enzyme probably by binding H115 at the active groove. This represents the first purified FA-o-MT from Hemiptera with preferred biological activity for FA and not JHA.
authors.affiliation: USDA-ARS, Ft. Pierce, FL 34945, USA.
authors.forename: Evelien
authors.initials: E
authors.lastname: Van Ekert
authors.affiliation: USDA-ARS, Ft. Pierce, FL 34945, USA.
authors.forename: Robert G
authors.initials: RG
authors.lastname: Shatters
authors.affiliation: Université de Toulouse, UPS, Institut de Recherche pour le Développement (IRD), UMR 152 Pharma-Dev, Université Toulouse 3, Faculté des Sciences Pharmaceutiques, F-31062 Toulouse cedex 09, France.
authors.forename: Pierre
authors.initials: P
authors.lastname: Rougé
authors.affiliation: Indian River Research and Education Center, University of Florida, FL 34945, USA.
authors.forename: Charles A
authors.initials: CA
authors.lastname: Powell
authors.affiliation: Universiteit Gent, Gent 9000, Belgium.
authors.forename: Guy
authors.initials: G
authors.lastname: Smagghe
authors.affiliation: USDA-ARS, Ft. Pierce, FL 34945, USA.
authors.forename: Dov
authors.initials: D
authors.lastname: Borovsky
keywords: 3D modeling; Diaphorina citri; FA, farnesoic acid; FA-o-MT, farnesoic acid o-methyltransferase; Farnesoic acid methyltransferase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Gene expression; JH, juvenile hormone; JHA, juvenile hormone acid; JHAMT, juvenile hormone acid methyltransferase; Juvenile hormone acid methyltransferase; MF, methyl farnesoate; MMLV, Moloney murine leukemia virus; RP, reversed phase; SAM, S-adenosyl methionine; SAM-MT, S-adenosyl methionine-dependent methyltransferase; fmtD, Diaphorina citri farnesoic acid o-methyltransferase gene; jmtD, juvenile hormone acid methyl transferase ortholog gene of Diaphorina citri