Aedes aegypti juvenile hormone acid methyl transferase, the ultimate enzyme in the biosynthetic pathway of juvenile hormone III, exhibits substrate control

作者：Van Ekert Evelien; Heylen Kevin; Rouge Pierre; Powell Charles A; Shatters Robert G Jr; Smagghe Guy; Borovsky Dov^*

来源：Journal of Insect Physiology, 2014, 64: 62-73.

DOI：10.1016/j.jinsphys.2014.03.001

• Corpora allata
• Aedes aegypti
• Ovary
• Juvenile hormone methyltransferase
• 3D Modeling
• Gene expression

We report on the cloning, sequencing, characterization, 3D modeling and docking of Aedes aegypti juvenile hormone acid methyl transferase (AeaJHAMT), the enzyme that converts juvenile hormone acid (JHA) into juvenile hormone (JH). Purified recombinant AeaJHAMT was extensively characterized for enzymatic activity and the Michaelis Menten kinetic parameters K-m, V-max, k(cat) (turn over number) and k(cat)/K-m, (catalytic efficiency) using JHA and its analogues as substrates. AeaJHAMT methylates JHA III 5-fold faster than farnesoic acid (FA). Significant differences in lower methyl transferase (MT) activities towards the cis/trans/trans, cis/trans/cis and the trans/cis/cis isomers of JHA I (1.32, 4.71 and 156-fold, respectively) indicate that substrate chirality is important for proper alignment at the catalytic cavity and for efficient methyl transfer by%26apos;S-adenosyl methionine (SAM). Our 3D model shows a potential binding site below the main catalytic cavity for JHA analogues causing conformational change and steric hindrance in the transfer of the methyl group to JHA III. These, in silico, observations were corroborated by, in vitro, studies showing that several JHA analogues are potent inhibitors of AeaJHAMT. In vitro, and in vivo studies using [H-3-methyl]SAM show that the enzyme is present and active throughout the adult life stage of A. aegypti. Tissue specific expressions of the JHAMT gene of A. aegypti (jmtA) transcript during the life cycle of A. aegypti show that AeaJHAMT is a constitutive enzyme and jmtA transcript is expressed in the corpora allata (CA), and the ovary before and after the blood meal. These results indicate that JH III can be synthesized from JHA III by the mosquito ovary, suggesting that ovarian JH III may play an important physiological role in ovarian development and reproduction. Incubating AeaJHAMT with highly pure synthetic substrates indicates that JHA III is the enzyme%26apos;s preferred substrate, suggesting that AeaJHAMT is the ultimate enzyme in the biosynthetic pathway of JH III.

• 出版日期2014-5