Chapter 16
Microbiological Identification and Characterization of an Amino Acid Biosynthetic Enzyme as the Site
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of Sulfonylurea Herbicide Action Robert A. LaRossa, S. Carl Falco, Barbara J. Mazur, Kenneth J. Livak, John V. Schloss, Dana R. Smulski, Tina K. Van Dyk, and Narendra S. Yadav Central Research and Development Department, Experimental Station, Ε. I. duFont de Nemours and Company, Wilmington, DE 19898
The i n a b i l i t y to synthesize many amino acids d i s t i n guishes animal c e l l s from those of plants, fungi and bacteria. Potential h e r b i c i d a l targets include the plant enzymes responsible for synthesizing these amino acids since their selective i n h i b i t i o n might r e s u l t in control of weed growth and low animal t o x i c i t y . Microbiological studies have recently indicated that several herbicides interfere with amino acid biosynthesis. Elucidation of the enzymic target of a sulfonylurea herbicide, s u l f o meturon methyl (SM), i l l u s t r a t e s the utility of a microbial approach to studies of herbicide action. Specific reversal of SM-mediated i n h i b i t i o n of b a c t e r i a l growth by isoleucine, methionine and pantothenate f i r s t suggested that the s i t e of action was acetolactate syn thase (ALS), a branched chain amino acid biosynthetic enzyme. Results confirming this p r e d i c i t i o n include the potent i n v i t r o i n h i b i t i o n of microbial and plant ALS a c t i v i t y by SM, the mapping of both b a c t e r i a l and yeast SM-resistant mutations to genes encoding ALS and the altered, SM-resistant forms of ALS found i n microbial and plant mutants. Comparison of the DNA sequence of wild type and mutant s t r u c t u r a l genes encoding ALS from both bacteria and yeast has established the molecular bases for the herbicide-resistant phenotypes. The detailed understanding of branched chain amino acid biosynthesis i n microbes has thus provided both tools and insights into the mode of SM action.
Microbes are more amenable to p h y s i o l o g i c a l , biochemical and genetic manipulation than metazoans and metaphytes. Diverse problems ranging from c e l l u l a r d i f f e r e n t i a t i o n and morphogenesis to sensory adaptation and m u l t i c e l l u l a r development are currently being studied i n microorganisms (1)· A primary goal of these studies has been to develop a r e q u i s i t e genetic arsenal i n each organism with which to 0097-6156/87/0334-0190$06.00/0 © 1987 American Chemical Society
LeBaron et al.; Biotechnology in Agricultural Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
16.
LAROSSA E T A L .
Characterization of an Amino Acid Biosynthetic Enzyme191
probe these complex processes (2). Processes important to plants such as i n t e r c e l l u l a r communication occurring during development and photosynthesis are being approached i n microbes u t i l i z i n g both c l a s s i c a l and molecular genetics as well as biochemistry and physiology. Indeed, s i g n i f i c a n t progress i s being achieved (e. g. 2_
2). Another process of importance to plant science i s amino acid biosynthesis. Plants and most microbes share the capacity to synthesize the twenty common amino acids from c e n t r a l , key metabol i t e s (see Figure 1). In contrast animals must ingest ten amino acids " e s s e n t i a l " to their diet; they are unable to produce leucine, v a l i n e , isoleucine, threonine, methionine, l y s i n e , h i s t i d i n e , tryptophan, tyrosine and phenylalanine. A sufficiently specific chemical i n h i b i t i n g the biosynthesis of an essential amino acid thus might control weed growth and display l i t t l e t o x i c i t y towards mammals. Indeed a number of herbicides i n t e r f e r e with the biosynthesis of the essential amino acids (4^, see Table I ) . Table I. Herbicides Interfering with Essential Amino Acid Biosynthesis Herbicide
Pathway
Aminotriazole
Histidine
Glyphosate
Aromatic
Sulfometuron methyl Chlorsulfuron Imidazoline) ne s
Branched Chain Branched Chain Branched Chain
Enzyme Imidazole g l y c e r o l phosphate dehydratase Enolpyruv ylshikimate phosphate synthase Acetolactate synthase Acetolactate synthase Acetolactate synthase
adapted from 4^ Studies of amino acid biosynthesis i n plants have a great advantage i n comparison to work done on other important plant processes. Since amino acid metabolism has been extensively studied i n enteric bacteria (Salmonella typhimurium and Escherichia c o l i ) and a fungus (Saccharomyces cerevisiae) these organisms provide a convenient and powerful springboard for further i n v e s t i g a t i o n . The pathways were elucidated i n these organisms and have been extensively analyzed (5)· The r e q u i s i t e biochemical and genetic techniques have been developed i n these organisms and technological hurdles are thus few. Tools are r e a d i l y available including substrates, cofactors, s t r u c t u r a l genes, regulatory mutants, enzyme overproducing strains and protocols for enzyme p u r i f i c a t i o n . Detailed understanding of the stringent response (Figure 2, 60, a global regulatory c i r c u i t reorienting the bacterium's metabolism i n response to a l i m i t i n g supply of any amino acid (6^, 7) was quite useful i n elucidating the mode of sulfometuron methyl i n h i b i t i o n (see below). Upon amino acid l i m i t a t i o n the relA product produces pppGpp and ppGpp, two nucleotides that have been c a l l e d alarmones (7) since they signal a paucity of amino acids available for protein synthesis. These nucleotides stimulate expression of amino acid synthetic genes resulting i n elevated i n t r a c e l l u l a r l e v e l s of the enzymes
LeBaron et al.; Biotechnology in Agricultural Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
B I O T E C H N O L O G Y IN A G R I C U L T U R A L C H E M I S T R Y
192
responsible for amino acid synthesis. Thus relA mutants are defective i n this response and are thus more sensitive to a wide spectrum of amino acid antagonists than are wild type b a c t e r i a . Such relA mutants are therefore p a r t i c u l a r l y useful for analyzing i n h i b i t o r s of amino acid metabolism (7). Here we describe the i d e n t i f i c a t i o n of acetolactate synthase, a branched-chain amino acid biosynthetic enzyme (see Figure 3), as the target of the sulfonylurea herbicide, sulfometuron methyl (SM, Figure 4). Sulfometuron Methyl The broad-spectrum herbicide sulfometuron methyl i s a member of a class of sulfonylurea analogues being developed by the Du Pont Company (8). These herbicides are characterized by e x t r a o r d i n a r i l y low application rates of between 5 and 35 grams per hectare, short half l i v e s i n many s o i l s and low t o x i c i t y towards mammals (LDSO's > 0.5g/kg, 8). SM i s a broad spectrum herbicide while an analogue, chlorsulfuron (CS, figure 4), s e l e c t i v e l y i n t e r f e r e s with the growth of broad-leaf weeds while allowing wheat to proceed through i t s normal l i f e cycle (8)· The e a r l i e s t studies done on the mode of action of these two herbicides u t i l i z e d corn root t i p assays (9). Incorporation of the radioactive precursors thymidine, uridine and leucine into DNA, RNA and protein was examined. DNA synthesis, but not RNA and protein synthesis, was i n h i b i t e d by low l e v e l s of CS (9)· Despite an extensive search, the biochemical basis for this i n h i b i t i o n of DNA synthesis i s unknown (10)· Thus new approaches were taken. Genetic analyses of tobacco c e l l culture mutants resistant to SM and CS demonstrated that mutation of a single nuclear gene conferred a resistant phenotype upon tobacco but did not indicate a biochemical target. Since mutations selected i n c e l l culture cause resistance i n regenerated plants, i t was suspected that CS and SM might i n h i b i t a basic metabolic step common to a l l plant c e l l s (11). Microbiology of SM Action The a b i l i t y of SM to i n h i b i t a v a r i e t y of microorganisms on minimal and complete media was examined by a disc d i f f u s i o n assay performed on s o l i d medium (12). This simple and f a c i l e assay allows a wide range of i n h i b i t o r concentrations to be examined on a single p e t r i dish. Both an Acinetobacter species and Citrobacter f r e u n d i i are i n h i b i t e d by SM on minimal medium; neither b a c t e r i a l species i s i n h i b i t e d on r i c h media (12) · The observation that SM-mediated i n h i b i t i o n of freundii i s enhanced upon supplementation of the minimal medium by valine (12) suggested a means to achieve SM i n h i b i t i o n of the g e n e t i c a l l y tractable S^. typhimurium by synergism of the herbicide with the amino a c i d . Indeed neither valine nor SM alone i n h i b i t wild type JS. typhimurium; however i n combination the two chemicals severely retard the growth of this bacterium (Table I I , 12).
LeBaron et al.; Biotechnology in Agricultural Chemistry ACS Symposium Series; American Chemical Society: Washington, DC, 1987.
LAROSSA E T A L .
Characterization of an Amino Acid Biosynthetic Enzyme193
ARG
PRO
GLN
ASN
M X GLU
ASP «kg
-LYS
οαα
- MET
GLYτ
-ILE
THR'SER-
CYS
•VAL
pyr-
• 3-pg pep + e-4-p
atp + prpp
"ALA
LEU
N
TRP-
HIS TYR
PHE
Figure 1. An overview of amino acid biosynthesis. Central metabolites are written i n lower case; the amino acids e s s e n t i a l to the mammalian diet are underlined.
pppG ρ relA, uncharged tRNAôn ribosome
, pppA VpA pppGpp
Y
> turn off of tRNA and ribosomal RNA genes Figure 2.
threonine Τ TD a-ketobutyrate [CH CH C0C00H] 3
ppGpp
