LABORATORY OF BIOCHEMICAL ECOLOGY,

University of Kentucky,Lexington, KY 40506

e-mail: garose@pop.uky.edu  

INTRODUCTION

Higher plants produce a large number of compounds which are not part of the repretoire of essential metabolites. These natural products comprise the "secondary metabolites" of higher plants. Many toxic substances, including a number of nonprotein amino acids, are part of this assemblage; they constitute an important group of compounds which function in plant defense against herbivores, pests, predators and pathogens. These protective compounds or allelochemicals play a critical role in organismic interactions involving plants; yet, only in limited instances do we understand their mode of action at the biochemical level. This deficiency has motivated my career-long interest in L-canavanine, a nonprotein amino acid synthesized by leguminous plants that is a potent L-arginine antimetabolite.

L-Canavanine is synthesized only by leguminous plants which are members of a large family of vascular plants known as the Fabaceae (Leguminosae). While canavanine can be found in all tissues of the living plant, it is stored primarily in the seeds where it serves to protect this organ from many potential consumers-particularly insects and to provide nitrogen for the developing embryo.

CANAVANINE CHEMISTRY

Examination of these two amino acids reveals some interesting similarities and differences. Overall, the molecules are remarkably similar, but the terminal methylene group of the guanidino moiety of arginine is replaced by oxygen to create a novel functionality-a guanidinooxy group. The pK_a of the guanidino group is 12.48 as compared to 7.04 for the guanidinooxy moiety. This property reflects the fact that canavanine is a far more acidic amino acid than is arginine. Under physiological conditions, arginine is fully protonated and positively charged. Canavanine, in contrast, gives up a proton more readily than arginine and is therefore not fully charged at these conditions.

CANAVANINE-CONTAINING PROTEINS

Because of its structural similarity to arginine, canavanine is a substrate for the enzyme, arginyl-tRNA synthetase. This protein is responsible for placing arginine into the nascent protein.

CANAVANINE AS A CHEMOTHERAPEUTIC AGENT

• Canavanine exhibits significant antineoplastic activity against rat colonic cancer in vivo.
• The antineoplastic activity of this nonprotein amino acids has been demonstrated in numberous in vitro studies involving a number of human pancreatic cancer cell lines, particularly MIAPaCa-2.

INSECT-BASED CANAVANINE STUDIES

• Investigations of the neotropic bruchid beetle, Caryedes brasiliensis, [Bruchidae] the sole predator of the canavanine-laden seeds of the neotropical legume, Dioclea megacarpa [Fabaceae].
• Elucidation of the biochemical basis for the remarkable tolerance of the tobacco budworm, Heliothis virescens [Noctuidae] to canavanine.
• Probing the biochemical basis for canavanine's antimetabolic properties in insects.

PLANT-BASED CANAVANINE STUDIES  

Investigations of canavanine catabolism in the jack bean, Canavalia ensiformis [Fabaceae] have provided an in-depth picture of how the nitrogen atoms of the guanidinooxy group of canavanine are mobilized for subsequent use by the developing plant.

CANAVANINE CHEMISTRY

• Synthesis of:
• L-[¹⁴C-guanidinooxy]canavanine
• L-[¹⁴C-U]canavanine
• ¹⁵N-labeled canavanine
• Isolation of canavanine from acetone-defatted jack bean, Canavalia ensiformis seeds.

L-CANALINE

Enzymatic hydrolysis of L-canavanine by arginase (EC 3.1.2.5) hydrolytically cleaves this molecule to yield urea and a novel nonprotein amino acid L-canaline that bears structurally analogy to L-ornithine. L-Canaline, L-2-amino-4-(aminooxy)butyric acid, is unique in being the only naturally occurring amino acid possessing an aminooxy moiety:

This nonprotein amino acid reacts vigorously with the pyridoxal phosphate moiety of vitamin B6-containing enzymes to form a covalently-bound oxime that inactivates, often irreversibly, the enzyme. As such it is a powerful antimetabolite that is formed in any canavanine-containing legume since arginase is distributed universally in this family of higher plants. Recently, this natural product was shown to exhibit significant antineoplastic activity against MIAPaCa-2, a human pancreatic cancer cell line. Additional information on this interesting nonprotein amino acid is available.