REGULATION OF L-ISOLEUCINE BIOSYNTHESIS IN THE PHOTOSYNTHETIC BACTERIUM RHODOSPIRILLUM RUBRUM* BY CATHERINE NINGt AND HOWARD GESTt

ADOLPHUS BUSCH III LABORATORY OF MOLECULAR BIOLOGY, WASHINGTON UNIVERSITY, ST. LOUIS, MISSOURI Communicated by Martin D. Kamen, October 17, 1966 Biosynthetic threonine deaminase (TD) catalyzes the conversion of L-threonine to a-ketobutyrate and is the first of a series of enzymes uniquely concerned with the synthesis of L-isoleucine in bacteria. Partial control of isoleucine production in such cells is provided through feedback inhibition of the deaminase by the end product, isoleucine.' The specific feedback properties of threonine deaminases, however, may differ significantly in different microorganisms. Thus, isoleucine is a potent inhibitor of the cell-free enzyme from Escherichia coli,2 Salmonella typhimu- rium,3 and Rhodopseudomonas capsulatus,4 even in the presence of relatively high threonine concentrations. Activity of the Rhodospirillum rubrum (strain Si) en- zyme, on the other hand, is appreciably inhibited by isoleucine only at low threonine concentrations, and the inhibition is gradually relieved as the substrate concentra- tion is increased.4 Although the in vitro behavior of the enzyme could be considered to reflect "loose" or inefficient feedback control of TD activity in R. rubrum,4 other interpretations are possible. This communication presents a re-evaluation of the feedback control pat- tern for synthesis of L-isoleucine and other amino acids of the aspartic family (threonine, methionine, and lysine) in this organism based, in part, on further study of the "native" TD and of an experimentally modified form with increased sensitiv- ity to feedback inhibition by isoleucine. Experimental.-The source of TD for the experiments reported here was a mutant strain of R. rubrum Si; the mutant, designated as S1H, was isolated from the cell population which even- tually developed in an anaerobic illuminated culture, in a medium containing 0.4% Lserine and 0.016% glycine as the only organic substrates. The mutant grows readily in malate plus am- monium salt media and shows a greatly elevated TD content as compared to the wild-type strain (approx. 15- to 20-fold). With glutamate as the nitrogen source, growth of the parent strain is markedly inhibited in the presence of exogenous L-threonine5 and as might be expected, growth of the mutant is much less susceptible to inhibition by this amino acid. Extracts were prepared from cells grown photosynthetically, under anaerobic conditions, in the synthetic malate + ammonium sulfate (1.25 gm/liter) medium described by Ormerod et al.' Since the TD of R. rubrum is unstable at 0-5°C under certain conditions,4 cells were harvested and all subsequent operations conducted at room temperature (unless otherwise noted). Follow- ing a single wash with 0.5 M potassium phosphate buffer pH 8.0 containing pyridoxal phosphate (25 ug/ml) and a low concentration of I-isoleucine (0.1 mM), the cells were disrupted by grinding with fine alumina according to the procedure of McIlwain.7 The ground mixture was extracted with 5 ml of the buffer for each gram of cell paste used. Alumina and cell debris were removed bylow-speed centrifugation, and the extract wasfurtherclarified bya second centrifugation at 65,000 X g for 4 hr (Spinco rotor no. 30). The extract was then heated at 600C for 10 min and coagulated proteinswereremoved bycentrifugation. Saturated ammonium sulfate solution, adjusted to pH 7.4 with concentrated ammonium hydroxide, was added dropwise to the heated fluid until 0.35 satura- tion was attained. The resulting precipitate was collected by centrifugation, triturated in the buffer noted to give a protein concentration of 5-10 mg/ml, and the preparation dialyzed for approximately 2 hr against a relatively large volume of buffer. At this stage, the enzyme was reasonably stable to storage at -15°C, about 50% of the activity remaining after 1 week. Ordi- 1823 Downloaded by guest on September 30, 2021 1824 BIOCHEMISTRY: NING AND GEST PROC. N. A. S.

narily, the extent of purification was 30- to 40-fold. Papain-treated TD for the experiments of Figures 1 and 3 was prepared as follows: The deaminase, partially purified as described, was incubated with crystalline papain in a malonic acid (pH 6.5) + ,3-mercaptoethanol buffer (see details in legends) for 5-10 min at room temperature. TD was then separated from the papain by filtration of the mixture through a small Sephadex G-75 column (equilibrated with 0.05 M potassium phosphate buffer pH 8.0); additional 0.05 M buffer was added to the column, as necessary, to displace the deaminase. For the kinetic experiment of Figure 2, the column separa- tion was not employed. Deaminase activity was estimated by a modification of the procedure of Friedemann and Haugen.8 The reaction mixtures contained the following components, in a final volume of 1 ml: potassium phosphate pH 8.0, 100 Mmoles; pyridoxal phosphate, 5 sg; L-threonine, enzyme, and other additions as indicated. Following incubation for 30 min at 370C, the reaction was termi- nated by adding 0.3 ml of a 2: 1 mixture (v/v) of 0.2% dinitrophenylhydrazine hydrochloride (in 2 N HCl) and 30% trichloroacetic acid. After additional incubation for 10 min, 1 ml of 2.5 N NaOH was added. Ten min were then allowed for color development before measuring absor- bancy, at 540 my, in a Zeiss PMQ II spectrophotometer (1-cm light path). Blank reaction mix- tures contained all relevant components, except substrate. Enzyme activity is expressed in terms of absorbancy; a value of 1.0 is equivalent to 0.25 Mumole of a-ketobutyrate. Results and Discussion.-Feedback inhibition of the "native" S1H threonine deami- nase by isoleucine: The effect of isoleucine on activity of the "native" R. rubrum S1H deaminase at various substrate concentrations is illustrated by data in Figure 1 (solid lines). With 1 mM isoleucine, significant inhibition occurs only at threonine concentrations less than 3 mM. At higher isoleucine concentrations (e.g., 10 mM), the same general relationships of the curves (4± isoleucine) are observed, as reported earlier for the enzyme of the parent S1 strain.4 The sigmoidal character of the sub- strate-dependence curve, indicating cooperative effects frequently seen with regula- tory enzymes, is considerably exaggerated in the presence of isoleucine. Effect of papain on the S1H threonine deaminase: Taketa and Pogell9 have demon- strated that the regulatory properties of mammalian fructose-1,6-diphosphatase can be markedly altered by treatment of the enzyme with papain. The proteolytic enzyme progressively inactivates the diphosphatase, but appears to have a preferen- tial effect on binding sites for an allosteric effector, adenosine 5'-monophosphate. By appropriate exposure to papain, active diphosphatase which is no longer in- hibited by the effector can be obtained. Similar treatment of the R. rubrum S1H threonine deaminase results in the opposite effect, i.e., increase in sensitivity to the effector, isoleucine. This is shown in Figure 2 (see also Fig. 1). As the deaminase becomes inactivated, there is a rapid gain in feedback sensitivity to isoleucine at high substrate concentration (10 mM threonine in Fig. 2). Inactiva-

1.2- PAPAIN TREATED FIG. 1.-Feedback inhibition of R. ru- 1.0 brum S1H threonine deaminase activity >_ _ by L-isoleucine (IL). For the "native" F 0.8 _ enzyme curves (solid lines), the reaction P / mixtures each contained 13 Aig of protein; LU OA + lmM IL --A threonine and isoleucine were added at the Z __ concentrations indicated. Papain-treated was from LUZ 0.4 / Z __theenzymesame(dashedammoniumlines)sulfatepreparedfraction, using

0.20.2 0.04 mg papain/mg bacterial protein (in _ 0.2 M malonic acid + 0.07 M fl-mercapto- o I1. ethanol buffer); each assay mixture con- 0 1.0 2.0 3.0 4.0 tained 46 ,ug of protein. L-THREONINE CONC. (mM) Downloaded by guest on September 30, 2021 VOL. 56, 1966 BIOCHEMISTRY: NING AND GEST 1825

FIG. 2.-Time course of papain inactiva- 9J tion and simultaneous change in feedback \ ACTIVITY sensitivity of R. rubrum SLH threonine 80 deaminase to L-isoleucine. The deaminase 70 D (same ammonium sulfate fraction as used for X \ was incubated with at C in 60 Fig. 1) papain 370 ac 0.15 M malonic acid + 0.1 M ft-mercapto- * ethanol buffer (bacterial protein, 2 mg/ml; 50XINHIBITION* papain, 0.14 mg/ml). At 1-min-intervals, a 40 -\0.1-ml sample was diluted 100-fold in 0.5 M >_ @ < >potassium phosphate buffer (pH 8.0), and the I- 30 activity of 0.5 ml of the dilution immediately U. 2o assayed using 10mM L-threonine as substrate in the absence and presence of L-isoleucine ,, lo (IL, 20 mM). In this experiment, 100% ac- 0 (MI,VME ,tivity corresponded to an absorbancy of 0.86 01 2 3 4 S 6 in the assay. TIME (MIN) tion of the TD beyond a given point can be avoided by removing the papain, which can be accomplished by filtration of the incubation mixture through Sephadex G-75 (the mol wt of papain is considerably smaller than that of the deaminase). This procedure was used for the experiment of Figure 1 (dashed lines), and for Figure 3, which compares the sensitivities of "native" and papain-treated enzymes in respect to feedback inhibition by various concentrations of isoleucine at a constant and high level of threonine (10 mM). These results indicate that papain can modify the S1H deaminase to a form which resembles the more feedback-sensitive enzymes observed in a number of other bacteria. The action of papain apparently does not cause gross changes in molecu- lar size of the enzyme, as evidenced by the fact that significant differences could not be detected in sucrose density gradient centrifugation patterns of the two "forms." Treatment of the R. rubrum S1 deaminase

with papain led to similar changes in sen- 1.0 0 sitivity of the enzyme to feedback inhibi- 09 tion by isoleucine. Indeed, examination of a number of general properties of the S1i .8 and S1H enzymes has not revealed sub-a stantial differences between them. OA Thefeedback control pattern in R. rubrum: M The control networks regulating exten- z. sively branched biosynthetic pathways in APAIN TREATED growing cells might well be expected to 0-4 _ have a high order of complexity, and this 0_3_E ___ll __lllll 8 10 12 14 16 20 is clearly indicatedyby numerous recent in- 0 2 4 6 LISOLEUCINE ~~~~~~~~~~~~~~~~~~~~CONIC.(mM)(m vestigations. Noteworthy in this con- nection are the pathway responsible for FIG. 3.-Feedback inhibition of "native" and papain-treated R. rubrum S1H threonine synthesis of the aspartic family amino deaminase by L-isoleucine at high substrate acids and that for production of aromatic concentration (10 mM Lthreonine). For the "native" enzyme curve, each reaction mixture compounds (phenylalanine, tyrosine, tryp- contained 6 pg of protein. Papain-treated tophan, etc.) in bacteria. For each path- enzyme was prepared using 0.08 mg papain/ mg bacterial protein (in 0.15 M malonic way, studies with different microorganisms acid + 0.1 M buffer); have revealed the existence of alternative each assay mixturefl-mercaptoethanolcontained 24 ug of protein. Downloaded by guest on September 30, 2021 1826 BIOCHEMISTRY: NING AND GEST PROC. N. A. S.

control patterns, designed to achieve economic and balanced production of the di- verse end products from common precursors.'0-'3 We assume that each master plan requires, or dictates, particular feedback control properties for key regulatory enzymes (e.g., those catalyzing the initial step and reactions at branch points), and that these are necessarily interdependent. Accordingly, the control properties of the TD of R. rubrum must be considered in relation to the over-all scheme for regulation of the aspartic family pathway in this bacterium. The regulatory network involves several unusual features thus far not observed in other organisms, but not likely to be uniquely found in R. rubrum. Of special significance is the fact that both L-threonine and L-isoleucine (which is pro- duced from threonine through a nonbranched sequence of reactions) have the ca- pacity of modulating the activity of at least three enzymes participating in synthesis of these amino acids. R. rubrum appears to have only one j-aspartokinase (initial enzyme), which is feedback-inhibited by threonine; isoleucine reverses the inhibi- tion, and in the absence of threonine is an activator of the enzyme.'0 The homo- serine dehydrogenase (third enzyme) behaves in a generally similar fashion. 14 Homoserine kinase, the next enzyme in the sequence leading to threonine and isoleucine, catalyzes phosphorylation of the branch-point compound L-homoserine to O-phospho-L-homoserine; its activity is subject to feedback inhibition by both threonine and isoleucine (the cell-free enzyme of R. rubrum, whentested at 250C, was found to have feedback properties resembling those of the E. colil" enzyme). It would seem that an important aspect of the master control plan in R. rubrum is deli- cate regulation of the concentration of threonine, which is an unusual compound in the sense that it is both an "intermediate" in isoleucine production and an "end product" for protein synthesis. Poising of the threonine concentration would of course require, in part, control of the rate of TD activity. Such control, through feedback inhibition by isoleucine, would in turn also facilitate regulation of isoleucine production. We regard the sensitivity of "early" enzymes of the pathway to iso- leucine as an indication that the isoleucine pool size, as well as that of threonine, must be under strict control. The data at hand suggest that as the intracellular concentration of threonine rises above a critical point, the complex feedback system begins to operate in the follow- ing way. Further synthesis of threonine is decelerated by feedback inhibition (by threonine) of fl-aspartokinase, homoserine dehydrogenase, and homoserine kinase activities, i.e., by slowing both production and phosphorylation of homoserine. Inhibition of the former two enzymes diminishes the rate of production of common intermediates also required in methionine and lysine synthesis. Conversion of threonine to isoleucine proceeds under feedback control by isoleucine (i.e., at the TD step). The isoleucine/threonine ratio thereby gradually increases. Since feedback inhibition of ,B-aspartokinase and the dehydrogenase by threonine is reversed by iso- leucine, an increasing ratio of isoleucine/threonine appears to be the "signal" for accelerated production of the common intermediates necessary for methionine, lysine, and threonine formation.'0 A number of additional controls presumably must also operate to ensure balanced production of the aspartic family and related amino acids, e.g., feedback effects by L-valine and L-leucine would be anticipated since, as indicated by studies'6 with other Gram-negative bacteria, several enzymes concerned with biosynthesis of these two amino acids also function as catalysts in the threonine isoleucine branch. Downloaded by guest on September 30, 2021 VOL. 56, 1966 BIOCHEMISTRY: NING AND GEST 1827

The foregoing considerations imply that through interlocking feedback (of enzyme activity) and repression controls, the intracellular concentration of threonine in growing R. rubrum cells is maintained within a range permitting effective feedback regulation of TD activity by isoleucine. Presumably, this range would correspond with a zone of threonine concentrations, set by the master "blueprint," over which the entire biosynthetic pathway is tuned for optimal function as well as maximal economy and "buffering" capacity. In this interpretation, complete reversal of the feedback inhibition of TD by "high" concentrations of threonine-as seen with the "native" cell-free enzyme-may be viewed as a potentiality which is probably never realized physiologically. The ability of papain to alter this potentiality of the R. rubrum enzyme (which is much more pronounced than with other bacterial threonine deaminases in vitro) and also the control properties of fructose-1,6-diphosphatase suggests that this and other proteolytic enzymes may prove to be particularly useful tools for exploring the special structural aspects of regulatory enzymes. Summary.-The cell-free biosynthetic threonine deaminase of Rhodospirillum rubrum superficially appears to be atypical in respect to feedback control in that in- hibition of enzyme activity by L-isoleucine (observed at low L-threonine concentra- tions) is relieved by increase of the substrate concentration to seemingly moderate levels. By appropriate treatment with papain, the deaminase can be modified to a form which shows significantly increased sensitivity to isoleucine. The present findings and other considerations indicate that, in vivo, isoleucine must be an effec- tive regulator of threonine deaminase activity; a relevant reinterpretation of the integrated feedback controls concerned with regulating synthesis of isoleucine and other amino acids of the aspartic family pathway in R. rubrum is presented. * This work was supported in part by grant AI-02640 from the National Institutes of Health and grant GB-1709 from the National Science Foundation. We are indebted to Dr. Maysie Hughes, Department of Pharmacology, St. Louis University School of Medicine, who isolated the R. rubrum S1H mutant while working in this laboratory; we thank Catherine Brenneman and Joyce Henry for expert technical assistance. f Aided by a grant for a postdoctoral fellowship from the American Cancer Society. Present address: Department of Microbiology, Washington University School of Medicine, St. Louis, Missouri. t Present address: Department of Microbiology, Indiana University, Bloomington, Indiana. I Moyed, H. S., and H. E. Umbarger, Physiol. Rev., 42, 444 (1962). 2 Umbarger, H. E., and B. Brown, J. Bacteriol., 73, 105 (1957). 3 Freundlich, M., and H. E. Umbarger, in Cold Spring Harbor Symposia on Quantitative Biology, vol. 28 (1963), p. 505. 4Hughes, M., C. Brenneman, and H. Gest, J. Bacteriol., 88, 1201 (1964). 6 Sturani, E., P. Datta, M. Hughes, and H. Gest, Science, 141, 1053 (1963). 6 Ormerod, J. G., K. S. Ormerod, and H. Gest, Arch. Biochem. Biophys., 94, 449 (1961). 7 McIlwain, H., J. Gen. Microbiol., 2, 288 (1948). 8 Friedemann, T. E., and G. E. Haugen, J. Biol. Chem., 147, 415 (1943). 9 Taketa, K., and B. M. Pogell, J. Biol. Chem., 240, 651 (1965). 10 Datta, P., and H. Gest, Nature, 203, 1259 (1964). 11 Datta, P., and H. Gest, these PROCEEDINGS, 52, 1004 (1964). 12Jensen, R. A., and E. W. Nester, J. Mol. Biol., 12, 468 (1965). 13 Stadtman, E. R., Bacteriol. Rev., 27, 170 (1963). 14 Datta, P., and H. Gest, J. Biol. Chem., 240, 3023 (1965). 15 Wormser, E. H., and A. B. Pardee, Arch. Biochem. Biophys., 78, 416 (1958). 16 Leavitt, R. I., and H. E. Umbarger, J. Biol. Chem., 236, 2486 (1961). Downloaded by guest on September 30, 2021