Characterization and inhibition of farnesyl diphosphate synthase

Corey Wayne Trobaugh, Purdue University

Abstract

Juvenile hormone (JH) is vital to normal insect growth and development. In the lepidopteran species of Manduca sexta, JH consists of five different methyl epoxy-farnesoate homologs, which are produced via the terpenoid biosynthetic pathway. Prenyltransferase, which catalyzes the formation of the JH carbon backbone, is a key enzyme of the pathway, since it may be partially responsible for control of JH homolog production. This research was concerned with the expression, purification and characterization of a dipteran and lepidopteran prenyltransferase, farnesyl diphosphate synthase (FPPS), and the design and synthesis of small molecules to selectively inhibit this enzyme. FPPS of the Drosophila melanogaster, a dipteran species, was cloned into the pET-28a(+) vector (Novagen) by our collaborators. The vector was transformed into E. coli BL21(λDE3) (Novagen) competent cells. The recombinant enzyme was grown in media containing 1% (w/v) glucose and expressed in media containing no glucose, at reduced temperatures (15°C), in the presence of 1 mM isopropyl β-D-thiogalactopyranoside (IPTG) and for an extended period of time (18 hr). The N-terminal his-tagged enzyme was liberated by sonication and purified with immobilized metal affinity chromatography utilizing Co2+ as the stationary metal ion. The purified enzyme required divalent metal for activity (Mg2+), and was activated by 0.1% Triton X-100. KM values of 31, 33, and 7 μM were determined for IPP, DMAPP and GPP respectively. A DMAPP homolog, HDMAPP, displayed a similar Michaelis constant of 38 μM. The design of moth specific inhibitors was accomplished by studying a 3-D homology model of the armyworm, P. unipuncta. Inhibitors were based on the bound product, FPP, and possible steric and electronic interactions within the active site of the enzyme. The first set of inhibitors is based on the catalytic mechanism of product formation. During diphosphate dissociation and chain elongation of the product, a resonance stabilized carbocation is formed. This carbocation species is mimicked by the use of a pyridinium moiety. The bisphosphonate group, a highly stable diphosphate mimic, is used to target the FPPS by forming a strong chelation interaction with the catalytic metal ions within the enzyme active site. Highly efficacious inhibitors were created by combing these two functionalities. To impart species selectivity, the pyridinium moiety was created using alkyl chains of varying length, i.e. methyl and ethyl. The second set of inhibitors was designed to be more species specific then the first and utilizes a π-π stacking interaction with the inhibitor and an active site residue. This set incorporates a second phenyl ring with varying degrees of electronic properties, namely, electron withdrawing and electron donating, to interact with a π system (histidine) in the active site. By incorporating these two types of electronic groups, these inhibitors allow for a full structure-activity relationship to be generated between the inhibitors and enzyme.

Degree

Ph.D.

Advisors

Sen, Purdue University.

Subject Area

Biochemistry|Organic chemistry

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