Inhibition of Acetolactate Synthase by ... - ACS Publications

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Inhibition of Acetolactate Synthase by Triazolopyrimidines A Review of Recent Developments Mani V. Subramanian and B. Clifford Gerwick Agricultural Products Research, Dow Chemical Company, Walnut Creek, CA 94598-0902 ALS is the f i r s t common enzyme in the biosynthetic route to valine, leucine and isoleucine. It is the site of action for the triazolopyrimidine (TP) herbicides as well as the sulfonylureas (SU) and imidazolinones (IM). These compounds act on the meristem and are slow to bring about plant death. Hence the opportunity to use metabolism of these herbicides to impart crop selectivity has been exploited successfully. Additions of valine, leucine, and isoleucine were found to relieve the growth inhibitory effects of TP on Bacillus cell cultures, soybean cell cultures, and Arabidopsis seedlings. ALS isolated from a number of sources was found to be sensitive to TP at nM levels. The barley enzyme has been amenable to purification. A purification procedure that gives >60 % recovery and 235-fold purification is described. The interaction of TP with ALS is non-covalent and the inhibition is of a mixed type with respect to pyruvate. Like IM and SU, TP is a slow, tight binding inhibitor with a much greater affinity for the steady state complex than the free enzyme. Mutants of tobacco and soybean resistant to TP have been isolated in tissue culture. Initial analysis suggests ALS in these mutants is desensitized to TP. Acetolactate synthase (ALS, EC 4.1.3.18) is the f i r s t common enzyme in the biosynthetic route to the branched chain amino acids, valine, leucine and isoleucine. It is the primary target site of action for at least three structurally distinct classes of herbicides, the imidazolinones (IM), sulfonylureas (SU), and triazolopyrimidines (TP) (Figure 1). SU and IM were discovered in greenhouse screening programs whereas TP was subsequently targeted as a herbicide. Numerous substitution patterns can be incorporated into the basic structure of a l l three classes of herbicides to provide crop selectivity as well as broad spectrum weed control. This is amply demonstrated in the seven products based on SU and four based on IM already in the market. A number of others are in various stages of development. The rapid success of ALS inhibitors as herbicidal products has attracted a world-wide research commitment. Not since the photosystem II c

0097-6156/89/0389-0277$06.00A) 1989 American Chemical Society

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i n h i b i t o r s (1), has a single mode of herbicide action had such p o t e n t i a l to reshape weed control chemistry and technology. SU and IM (Figure 1), are proprietary chemistry of DuPont (2.3) and American Cyanamid (4), respectively. The substituted 1,2,4-triazolo[l,5-a]pyrimidines (TP, Figure 1) are a new class of herbicides under development at Dow Chemical Co. (5). A number of reviews have already appeared on the biology and biochemistry of SU (6-10) and IM (11.12). Hence, the focus of this a r t i c l e has been on the work done at our premises on TP. Wherever appropriate, our r e s u l t s have been compared with those of SU and IM. Herbicidal Characteristics of TP The i n h i b i t i o n of the primary target, ALS, r e s u l t s i n a number of d i s t i n c t i v e whole plant symptoms. Hence, the h e r b i c i d a l biology of TP i s s t r i k i n g l y s i m i l a r to that exhibited by SU and IM. Growth of sensitive species i s retarded within a matter of hours of a p p l i c a t i o n although v i s i b l e e f f e c t s may not be observed for several days (for an account of observations with IM and SU see references 12 and 13, respectively). Symptoms appear f i r s t i n the upper meristematic regions of the plants as chlorosis and necrosis. The upper new leaves frequently take on a wilted appearance. The e f f e c t s then spread to the remaining parts of the plant. In many species including most legumes and the Panacoid grasses, there i s a c l e a r reddening of the midrib and l e a f veins. Complete desiccation of the plant may occur i n 7-10 days under ideal growing conditions but may take up to 6-8 weeks under lesser conditions. The actual l e t h a l event r e s u l t i n g from i n h i b i t i o n of ALS by TP, SU, or IM has been the subject of debate. Although the plant i s deprived of valine, leucine and isoleucine as a consequence of enzyme i n h i b i t i o n , protein synthesis continues at normal rates even a f t e r plant growth has begun to slow (12.13). Accumulation of one of the substrates of ALS, 2-ketobutyrate, has been implicated i n the cause of death i n microbes (9.14) . This substrate has also been shown to undergo transamination to the corresponding amino acid i n Lemna minor (15). However, the t o x i c i t y due to this keto acid has not been c l e a r l y demonstrated i n higher plants. One of the e a r l i e s t b i o l o g i c a l responses of SU treatment of plants i s the i n h i b i t i o n of c e l l d i v i s i o n (13). I t i s not clear how this i s related to ALS i n h i b i t i o n . While there i s consensus among workers that ALS i s the primary target s i t e for these herbicides, the actual biochemical cascades leading to death remain equivocal. Since TP, SU and IM are slow to bring about plant death, there are s i g n i f i c a n t opportunities to exploit metabolism of the herbicide to influence crop tolerance. Metabolism has indeed been the overr i d i n g parameter determining crop s e l e c t i v i t y (5c.16.17). ALS i n h i b i t i n g herbicides i n development and/or f u l l commercialization are known to have s e l e c t i v i t i e s to a l l the major crops including corn, soybeans, wheat, barley, r i c e , cotton and canola. Mode of Action of TP Several analogs of TP at 1500 ppm cause a growth lag of 25-50 hrs of E. c o l i (K-12) growing i n minimal media. The growth lag was comp l e t e l y abolished with the addition of casamino acids or a mixture of

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valine, leucine and isoleucine to the media. None of the other amino acids were e f f e c t i v e i n a l l e v i a t i n g the growth lag. The growth of B a c i l l u s s u b t i l i s was also completely i n h i b i t e d by TP but at concentrations as low as 80 ppm. Again, the growth i n h i b i t i o n was n u l l i f i e d upon addition of a l l the three branched chain amino acids (Figure 2). Both chlorsulfuron (CS), a SU and imazaquin, an IM, produced i d e n t i c a l e f f e c t s to TP on the above two bacteria. The difference i n the e f f e c t of these herbicides on the two bacteria can be attributed to the genetic regulation and isozyme patterns of ALS i n the two organisms. I t i s well documented that E. c o l i has an isozyme (ALS I) that has very low s e n s i t i v i t y to SU (18). Concentrations as low as 6 ppb of TP completely i n h i b i t e d soybean suspension cultures and Arabidopsis thaiiana. Consistent with the observations on b a c t e r i a l cultures, the presence of v a l i n e , leucine and isoleucine i n the media completely reversed the growth i n h i b i t i o n (Figures 3 & 4). In the case of Arabidopsis. the seedl i n g growth i n the presence of TP was proportional to the concent r a t i o n of the three amino acids i n the medium. A growth l e v e l equal to the control was achieved at an amino acid concentration of 0.35 mM (Figure 4). C o l l e c t i v e l y , the growth studies support that the primary mode of action of TP i n microorganisms and plants i s the disruption of branched chain amino a c i d biosynthesis. The v e r i f i c a t i o n of the mode of action of TP followed the l o g i c established i n studying SU (18-21) and IM (22-24). Although o r i g i n a l l y thought to i n h i b i t plant growth by arresting c e l l d i v i s i o n (13.25), subsequent experiments using b a c t e r i a (18), excised pea root t i p s (19) and pea seedlings (19) c l e a r l y proved that SU blocked the biosynthesis of the branched chain amino acids. Likewise, growth studies with corn cultures and corn plants revealed the mode of action of IM (22-24). I n h i b i t i o n of ALS bv TP The requirement for a l l three branched chain amino acids to n u l l i f y the i n h i b i t o r y e f f e c t s of TP suggested that the target enzyme i s ALS. ALS was i s o l a t e d from barley seedlings as a 0-33% Ammonium Sulfate p r e c i p i t a t e and examined for i n h i b i t i o n by TP. I t i s apparent from Figure 5 that the enzyme i s very sensitive to the compound. The 1(50) value (concentration required for 50% i n h i b i t i o n ) was calculated to be 0.047 uM. This value i s within the range reported for CS tested against ALS from d i f f e r e n t species (19). Imidazolinones are less potent with 1(50) values i n the range 2-12 uM (26). ALS i s o l a t e d from several species and t h e i r 1(50) values for TP i s shown i n Table I. Table I: I n h i b i t i o n of ALS from d i f f e r e n t species by TP Source of ALS Barley Arabidopsis Tobacco Cultures Cotton Cultures Soybean Cultures

1(50) uM 0.046 0.038 0.032 0.036 0.040

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Β

Figure 1. Three chemical families known to exhibit h e r b i c i d a l a c t i v i t y through the i n h i b i t i o n of acetolactate synthase. A. sulfonylurea (sulfometuron) B. imidazolinone (imazapyr) and C. A representative triazolopyrimidine.

2.000

TIME

(HRS.)

Figure 2. I n h i b i t i o n of growth of B a c i l l u s s u b t i l i s by t r i a z o l o pyrimidine; reversal by valine, leucine and isoleucine.

SUBRAMANIAN AND GERWICK

0

A 0

Figure 3 . cultures.

1 10

Inhibition of Acetolactate Synthase

1 20

1 1 30 40 TIME ( D A Y S )

1 50

60

E f f e c t of triazolopyrimidine on soybean suspension

Figure 4. E f f e c t of triazolopyrimidine on the growth of Arabidopsis thaiiana seedlings.

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ALS was assayed by quantifying the amount of acetoin formed from acetolactate (27). The enzyme from seedlings were assayed at pH 7.15 and that from cultures at 8.3. ALS from a number of higher plants has been readily extracted and assayed (19.22). However due to the i n s t a b i l i t y of the enzyme, there i s no e f f i c i e n t procedure for i t s p u r i f i c a t i o n . Recently, Muhitch et a l (28) described a four step p u r i f i c a t i o n procedure for ALS from maize suspension cultures. This method yielded enzyme preparation with high s p e c i f i c a c t i v i t y , but the recovery was low. We have examined ALS from d i f f e r e n t species i n order to i d e n t i f y the best source for p u r i f i c a t i o n and the results are given i n Table I I . Table I I :

ALS A c t i v i t i e s from Various Plant Sources

Specific A c t i v i t y (SA)

ALS Source

Crude Extract

Ammonium Sulfate ppt 35-50%

0-35% Soybean Cultures Tobacco Cultures Morningglory Cultures A l f a l f a Sprouts Mungbean Seedlings Barley Seedlings Cotton Cultures Arabidopsis Seedlings

865 1004

N.D 1121

87.8 420 10 50 60 8 N.D N.D

•N.D 289 420 1200 460 800

50 87 20 880 22

SA i n Table II - nmoles/hr/mg protein and N.D - not detectable. Tobacco, soy cultures and barley seedlings were the best source of ALS, both i n terms of s p e c i f i c a c t i v i t y and t o t a l u n i t s . The enzyme preparations from a l l sources were unstable i n buffer solutions i n spite of protective t h i o l agents. The i n a c t i v a t i o n of ALS i n the crude extract of tobacco showed a d i s t i n c t biphasic k i n e t i c s , implying the presence of at least two isozymes (unpubl i s h e d observations). The presence of two ALS genes i n tobacco (29) and at least three i n microorganisms (18) has also been noted by other workers. ALS from barley was most amenable to p u r i f i c a tion. Table III gives a p r o f i l e for the rapid p u r i f i c a t i o n of t h i s enzyme with high recovery. Table I I I :

Step

P u r i f i c a t i o n of ALS from Barley Seedlings

Volume (ml)

Crude Extract 620 0-33% AS ppt. 10 P2 Column (desalting) 14 Mono-Q-HPLC 2.2 Phenyl Agarose 2.1

Protein (mg/ml) 2.1 8.8 6.0 5.5 1.69

Total Units 91.1 106.8 117.6 82.32 58.8

S.A

0.07 1.213 1.40 6.8 16.6



In Table I I I , units have been defined as the amount of protein required f o r the formation of 1 umole product/min. The a c t i v i t y of the enzyme i n the crude extract was rather low accounting f o r an anomalous (>100 %) recovery i n the early stages of p u r i f i c a t i o n . The o v e r a l l recovery of the enzyme was >60 % with a 235-fold purification. The f i n a l preparation, however, was not homogenous. Barley has two isoforms of ALS which can be separated on a phenyl agarose column immediately after the ammonium sulfate p r e c i p i t a t i o n . One of the forms does not bind to the column and was too unstable to attempt p u r i f i c a t i o n . The d e t a i l s of p u r i f i c a t i o n i n Table I I I pertain to the f r a c t i o n with a f f i n i t y f o r phenyl agarose. The ALS isolated as described i n Table III displayed t y p i c a l Michaelis-Menten k i n e t i c s with respect to pyruvate with a Km of 2.44 mM. Substrate concentrations as high as 50x Km had no e f f e c t on the rate of the reaction. Thiamine pyrophosphate, FAD and Mg(2+) were an absolute requirement f o r c a t a l y s i s by the p u r i f i e d enzyme. These properties are consistent with observations made by others (30). Optimum a c t i v i t y was obtained at pH 7.1 and 37C, which were also the best conditions f o r i n h i b i t i o n by TP. There was no s i g n i f i c a n t difference i n the 1(50) value of TP whether ALS was taken a f t e r step 2 or 5, indicating low potential f o r non-specific binding of the herbicide to other proteins. Interaction of TP with ALS The interaction of a l l of the herbicides shown i n Figure 1 with ALS i s non-covalent (5b.28.30). R a d i o l a b e l e d TP can be quantitatively separated from barley ALS i n a gel f i l t r a t i o n column, with complete restoration of a c t i v i t y (data not shown). None of the three classes of herbicides bear any s t r u c t u r a l s i m i l a r i t y to the substrate or cofactors of ALS. This i s also r e f l e c t e d i n the k i n e t i c s of TP i n h i b i t i o n with respect to varying pyruvate, shown i n Figure 6. The pattern i s suggestive of a l i n e a r mixed type of i n h i b i t i o n . We have not examined the mode of i n h i b i t i o n with respect to varying thiamine pyrophosphate or FAD. SU have been reported to be a competitive i n h i b i t o r of ALS from Salmonella typhimurium (with respect to pyruvate, 30), and an uncompetitive i n h i b i t o r of the enzyme from Methanococcus (31). IM i s also an uncompetitive i n h i b i t o r of corn ALS (26). The time course of ALS i n h i b i t i o n by the three classes of compounds (Figure 1) i s d i s t i n c t l y biphasic (5b.28.30) with progressi v e l y decreasing enzyme a c t i v i t y . There i s at least a 5-10 f o l d difference between the i n i t i a l and the f i n a l steady-state 1(50) value (5b.18.28.30). TP, l i k e the SU and IM, are slow, t i g h t binding i n h i b i t o r s with a greater a f f i n i t y f o r the steady state complex of ALS than f o r the free enzyme (30). The interaction of SU and ALS II from S. typhimurium has been examined i n d e t a i l . Based on k i n e t i c s and spectroscopic evidence, Schloss et a l (30) have proposed that the i n h i b i t o r binds t i g h t l y to ALS-FAD-TPP (decarboxylated)-Mg(2+)pyruvate complex, at the s i t e of the second keto acid. Relationship between the binding s i t e s of TP. SU. and IM The s i m i l a r i t y i n the herbicide biology and biochemistry of the above s t r u c t u r a l l y d i s t i n c t compounds i s interesting as well as i n t r i g u i n g . One of the key questions i s whether these i n h i b i t o r s compete f o r the

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0.500

0.400

ο

0.300

m < 0.200

0.100 +

0.000 0.000

0.200

0.400 TP (ppm)

0.600

Figure 5. I n h i b i t i o n of barley acetolactate synthase by triazolopyrimidine.


Inhibition ofAcetolactate Synthase 285

same s i t e on ALS. In order to address t h i s question, we i s o l a t e d several mutants of tobacco, 10-100 f o l d r e s i s t a n t to growth on TP compared to the wild type (which succumbed at 6.5 ppb). Growth of a number of mutants was comparable to the control. I n i t i a l analyses of the mechanism of resistance revealed that most mutants had ALS desensitized to TP (new data, manuscript i n preparation). A few of them had susceptible enzyme but c l e a r l y tolerated higher concentrations of the compound. These are suspected to have enhanced metabolism of the herbicide. Among the 15 mutants examined, a l l were cross r e s i s t a n t to SU (chlorsulfuron) as well as IM (imazaquin). Many of these mutants remain to be characterized. However, our preliminary results suggest that a l l three classes of herbicides have highly overlapping binding (inhibitory) s i t e s on ALS. Cross resistance between SU and IM has already been documented by others (32) . Recently, Schloss et a l (33) showed that IM and TP were able to quantitatively displace a r a d i o l a b e l e d SU herbicide from ALS, i n d i c a t i n g competitive binding. Curiously, the SU ligand was also displaced by the quinone, Qo. I t was proposed that SU, TP, and IM bind to ALS i n a v e s t i g i a l quinone binding s i t e associated with the evolution of ALS from pyruvate oxidase. This enzyme i s an FAD-protein that catalyzes the oxidation of pyruvate to acetate. The reduced FAD i s regenerated v i a electron transfer to a quinone. Similarly, ALS i s a flavoprotein but i t s reaction (condensation of two keto acids) does not involve a net redox change. Schloss et a l (33) have suggested that these two enzymes share a common evolutionary heritage and that the quinone (herbicide) binding s i t e on ALS has been conserved due to the constraints of the c a t a l y t i c mechanism. While this i s an a t t r a c t i v e theory, the water soluble quinone Qo i s a r e l a t i v e l y weak i n h i b i t o r of barley ALS ( 1(50) - 30 uM i n our studies) compared to SU and TP. Further, the quinone, Q6, does not i n h i b i t barley ALS even at 200 uM. F i n a l l y , i t i s not known i f the three classes of herbicides i n h i b i t pyruvate oxidase. Recently, Shaner et a l (34) reported a mutant maize l i n e r e s i s t ant only to IM. S i m i l a r l y , McDevitt et a l (35) have also shown that s i t e directed substitution of d i f f e r e n t amino acids at f i v e d i f f e r e n t l o c i on the ALS sequence resulted i n s p e c i f i c resistance to SU herbicides. These studies suggest that IM and SU herbicides have non-overlapping binding domains on ALS. We have not examined the several TP r e s i s t a n t mutants f o r s p e c i f i c i t y of resistance. The genetics of SU resistance has been extensively studied. Mutants that are tolerant to SU have been i s o l a t e d i n b a c t e r i a (36), yeast (36), algae (37), haploid cultures of tobacco (38.39) as well as Arabidopsis (40). In a l l of the cases examined, ALS was found to be 100-fold less susceptible to the herbicide (36-40). compared to the wild type enzyme. A s i m i l a r change i n the s e n s i t i v i t y of ALS has been reported f o r IM r e s i s t a n t corn cultures (11). Plants regenerated from r e s i s t a n t tobacco cultures f u l l y retained the t r a i t , and transmitted i t as as a dominant or a semi-dominant nuclear gene (39). The molecular basis of SU resistance has been characterized i n microorganisms (36) and plants (41.42), by sequencing both the wild type and mutant ALS genes. Comparisons showed a single nucleotide difference that translated into one amino acid s u b s t i t u t i o n at the enzyme l e v e l . This change however, was not the same i n a l l r e s i s t a n t organisms. For example, i n E. c o l i . valine was substituted f o r alanine (36) and i n yeast, serine f o r proline (36). In tobacco,

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mutations i n both the ALS genes conferred resistance to SU (41). Introduction of r e s i s t a n t ALS gene into wild type resulted i n SU r e s i s t a n t transgenic plants (43). C o l l e c t i v e l y , the genetic studies o f f e r unequivocal evidence that ALS i s the sole primary target for the three classes of herbicides. Other applications for herbicide r e s i s t a n t mutants are imminent. SU r e s i s t a n t tobacco plants are expected i n f i e l d t r i a l s . The most advanced i n this new approach to crop s e l e c t i v i t y are IM r e s i s t a n t corn l i n e s . These plants are reportedly i n breeding t r i a l s with Pioneer Seed Co., to transfer the t r a i t into high y i e l d i n g hybrid l i n e s . Success i n this task w i l l not only expand market opportun i t i e s for commercial imidazolinones, but also allow corn production i n areas where imidazolinone carry-over may be a concern. A number of other crops engineered for herbicide resistance (either due to altered target s i t e (32.44), or as a r e s u l t of rapid herbicide metabolism (45), are at or near f i e l d t r i a l s (44.46). Engineering resistant crops i s quickly emerging as an alternative approach to achieving crop s e l e c t i v i t y . Another major application of herbicide resistance i s i t s u t i l i t y as a selectable marker. Like a n t i b i o t i c resistance i n b a c t e r i a l transformation, herbicide resistance should prove extremely useful for selecting transformants that are insect r e s i s t a n t , disease r e s i s t a n t , or engineered for other non-selectable t r a i t s . Many of these herbicide resistant markers have already been integrated into plant cloning vectors (32.43). Literature Cited 1. 2. 3.

4.

5.

6. 7. 8. 9.

Fedtke, C. in Herbicide Action; Springer Verlag: New York, 1982; Section C. Levitt, G. U.S. Patent 4,127,405, 1978. Sauers, F. and Levitt, G. In Pesticide Synthesis through Rational Approaches, Magee, P. S.; Kohn, G. K.; Menn, J. J., Eds.; ACS Symposium Series No 255; American Chemical Society: Washington, D. C., 1984; p. 21-28. Los, M. In Pesticide Science and Biotechnology, Greenhalgh, R.; Roberts, T. R., Eds.; Blackwell Scientific Publication: Oxford, 1987, p 35-42. (a) Kleschick, W. Α.; Costales, M. T.; Dunbar, J. E.; Meikle, R. W.; Monte, W. T.; Pearson, N. R.; Snider, S. W.; Vinogradoff, A. P., 194th ACS National Meeting, New Orleans, LA, September 3, 1987. AGRO 162. (b) Gerwick, B. C.; Loney, V.; Subramanian, M. V., 194th ACS National Meeting, New Orleans, LA, September 3, 1987. AGRO 163. (c) Hodges, C. C.; Avalos, J.; McCall, P. J.; Stafford, L. E.; Yackovitch, P. R., 194th ACS National Meeting, New Orleans, LA, September 3, 1987. AGRO 164. LaRossa, R.A. and Falco, S.C. Trends in Biotechnology. 1984; 2, pp 158-161. Ray, T. British Crop Protection Conference - Weeds. 1985; pp 131-138. Ray, T. Trends in Biotechnology. 1984; pp 180-183. LaRossa, R.A., Falco, S.C., Mazur, B.J., Livak. K.J. Schloss, J.V., Smulski, D.R., VanDyk, T.K. and Yadav, N.S. In ACS Symposium Series 334; 1987; pp 190-203.

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