Metabolism of pipecolic acid in a Pseudomonas species

ARCHIVES

OF

I3IOCHEMIRl~RY

Metabolism

AND

142, 32-39 (1971)

RIOPHYSICS

of Pipecolic

Acid

in a Pseudomonas

Species

VI. Precursors of Glutamate’ RICHARD Department

of Biochemistry,

A. HARTLINE University

AND

of California, California

Received

May

VICTOR

W. RODWELL

San Francisco Q&22

15, 1970; accepted

October

Medical

Center,

San Francisco,

12, 1970

The terminal metabolite in pipecolic acid catabolism by Pseudomonas putida is glutamic acid. The specific conversion of carbon G of pipecolate to carbon 5 of glutamate suggests that no symmetrical intermediates are involved. Cell extracts are capable of converting a-aminoadipate to glutamate.

acid (piperidine-2-carboxylic Pipecolic acid), a component of the free amino acid pool of certain legumes (l-3), is formed from lysine in plants (4, 5), Neurospora (6), and rat liver (7). Rat liver converts u-lysine to pipecolate (8) and L-lysine to saccharopine (8, 9). Pipecolate can (10) and saccharopine should readily form (Yaminoadipate, an established catabolite of lysine (11) and of pipecolate (10) in mammalian liver preparations. The initial reactions of L-pipecolate catabolism by Pseudomonas putida P2 (ATCC 25571) (12) (Fig. l), a soil microorganism that can grow on lysine or pipecolate as its sole source of carbon and nitrogen (13), were first shown in intact cells (13, 14) and later substantiated by purification and study of the enzymes-catalyzing reactions one (15) and three (16). Cultures growing on pipecolate excrete both a-aminoadipate and 1 This investigation was supported by grants from the National Science Foundation (G-18553) and the United States Public Health Service (GM-12048). Data are from the Ph.D. dissertation of Richard A. Hartline. For previous papers in this series, see Refs. 12-16. A preliminary report has appeared (34). e Present address: Department of Chemistry, Indiana University of Pennsylvania, Indiana, Pennsylvania 15701. 3 Present address: Department of Biochemistry, Purdue University, Lafayette, Indiana 47907. 32

glutamate (13). The cr-aminoadipate arises directly from pipecolate (13). The closely coordinated excretion of these two acids (13) suggested that glutamate might be a catabolite of pipecolate. We, therefore, investigated the metabolic origin of glutamate. MATERIALS

AND

METHODS

Chemicals from commercial sources included: DL-pipecolic acid (Aldrich Chemical Co.) ; DL-aaminoadipic acid and sodium pyruvate (Calbiothem); L-lysine and L-alanine (Merck and Co.); L-glutamic acid (Fisher Scientific Co.); L-aspartic acid (Distillation Products Industries) ; a-ketoadipic acid and zinc cr-hydroxyglutarate (K and K Laboratories, Inc.). Ascending thin-layer chromatography (TLC) was on 250-p cellulose or silica gel G layers, the latter activated 1 hr at 100” before use. Whatman 1 MM filter paper was used for descending paper chromatography. Preparative-scale electrophoresis was on Whatman 3 MM filter paper at pH 6.5 in pyridine:glacial acetic acid:water: : 1: 10:2QO (v/v) and at pH 3.5 in pyridine:glacial acetic acid:water::25:1:225 at 1500 V under Standard Thinner No. 325 (Standard Oil Co. of California) as coolant. For quantitative analysis of a-aminoadipate or glutamate, electrophoretograms on Whatman 1 MM paper were dipped in 0.5y0 ninhydrin in acetone, air-dried, heated at 70” for 30 min, and the purple glutamate or a-aminoadipate areas eluted with 1.5 ml of 20y0 ethanol containing 50 pg CuS04.5Hz0 per ml (17). Absorbance at 570 nm was measured against a reagent blank using a

GLCTSRIATE

L- PIPECOLATE

L-A’-PIPERIDEINE -6-CARBOXYLATE

FORMATION

FROM

L-ti-AMINOADIPATE & -SEMIALDEHYDE

PIPECOLATE

L-a-AMINOADIPATE

FIG. 1. Intermediates in L-pipecolate catabolism t,o L-ol-aminoadipate in P. p&da P2. Reaction one is catalyzed by L-pipecolate dehydrogenase (15) and reaction three by L-CEaminoadipate &semialdehyde:NAD oxidoreductase (16). Reaction two has not been studied in detail but may not be enzyme-catalyzed since L-A’-piperideine-6-carboxylate and Liu-aminoadipate &semialdehpde readily equilibrate at neutral pH (14). l-cm-path cuvette. Standard curves prepared by electrophoresis and elution of known quantities were linear from 0.1-0.6 kmoles of dicarboxylic acid. One-half mole of glutamate or ol-aminoadipate gave an absorbance of 0.25. Radioactive substrates were purified before use. nL-pipecolic acid-6J4C was absorbed to a 1 X lo-cm column of Dowex 50-X8 (H+), washed in with 100 ml HzO, and eluted by a nonlinear HCl gradient (2 ml fractions; 0.5 ml/min). The reservoir contained 2 N HCl and the mixing chamber 25 ml H20. Pipecolic acid (sp act 13,300 dps per pmole) emerging at 94-104 ml of HCl eluate was free of radioimpurities as judged by long-term autoradiography after two-dimensional cellulose TLC in 72y0 phenol followed by n-butanol:propionic acid:HzO: :92:47:61 (v/v). Commercial nr,-CZaminoadipic acid-6-W, which contained numerous radioimpurities, was purified by successive application of column chromatography on Dowex l-X8 (acetate) (13), preparative paper chromatography in n-butanol:propionic acid:H*O: :92:47:61 (v/v), preparative electrophoresis at pH 3.5, and column chromatography on Dowex l-X8 (formate) (18). The final product still contained traces of two radioimpurities, one of which probably was 01aminoadipic acid lactam (19). Carbon 5 of isolated radioactive glutamate was removed as COZ with hydrazoic acid. A doublesidearm vessel contained radioactive glutamic acid in 0.5 ml 100yc H2S04 prepared just before use by adding 1.3 ml fuming H*SOa (30y0 excess SO,) to 10 ml concentrated (96%) HzSOI. The upper sidearm well contained 0.4 ml 40% KOH. The lower sidearm was attached to a tube containing recrystallized NaN3. Decarboxylation was initiated by adding 20 mg NaN3 and allowed to proceed for 4 hr at 50” with addition of 10 mg NaN3 at hourly intervals. The reaction vessel was then cooled, flushed with Nz, and the gas bubbled through 0.1 N NaOH. COZ trapped in the upper sidearm and in the NaOH was converted to BaC03 which was removed by centrifugation, washed with ethanol, dried to constant weight, and

counted. The residual reaction mixture was diluted with 2.0 ml HzO, made basic with Ba(OHj2 and precipitated BaS04 removed by centrifugation. The basic supernatant liquid was applied to a 1 X IO-cm column of Dowex l-X8 (OH-), washed in with 100 ml HzO, and the column eluted with 100 ml 0.5 N acetic acid. The eluate was concentrated and subjected to preparative-scale paper electrophoresis at pH 3.5 to separate 2,4-diaminobutyrate from unreacted glutamate. Radioactive 2,4diaminobutyrat,e was then eluted and counted. Carbon 1 of isolated radioactive glutamate was removed as CO2 by oxidation with chromic acid (21) in a closed, t,hree-necked flask equipped with a reflux condenser and a Thunberg tube sidearm containing 2.0 ml 40y0 KOH. The flask contained radioactive glutamate plus 150 mg carrier glutamate dissolved in 1.0 ml of 14 N sulfuric acid. Oxidation was begun by addition of 0.6 ml of 35c0 aqueous solution of sodmm dichromate solution and allowed to proceed for 7 hr on a steam bath. The flask was then cooled, flushed with Nz, and BaCOa was isolated as described above. The residual reaction mixture was diluted with 20 ml HzO, neutralized, applied to a 1 X lo-cm column of Dowex l-X8 (OH-) and washed in with 75 ml H20. Elution was with 80 ml 0.5 N acetic acid. Succinic acid and unreacted glutamic acid in the acidic eluate were separated by preparative-scale TLC on silica gel G in benzene:methanol:acetic acid: : 45:45:8 (v/v). The succinic acid, visualized with bromcresol green, was scraped from the plate, eluted wit,h HzO, and counted. Radioactive materials were counted on planchets in an end-window gas-flow counter. Data were corrected for self-absorption to infinite thickness with the aid of either a BaC03 or a sodium succinate self-absorption curve. ANSCO nonscreen X-ray film was used for autoradiography. Preparation of ionic medium and conditions for growth of P. putida P2 have been described previously (12). Sole sources of carbon and nitrogen used for growth included nL-pipecolate, ammonium oL-malate, and nI,-or-aminoadipate,

34

HARTLINE

AND

L-lysine present initially at 25 mM concentration. Terms such as “pipecolate-grown cells,” etc., mean that the culture was grown on the indicated compound as sole source of carbon and nitrogen. Sonic extracts were prepared by treating a 2% (dry weight to volume) cell suspension in ionic medium for 1 hr in a 9 kc Raytheon sonic oscillator. Protein was determined by the method of Waddell

cw RESULTS

Isotope distribution in glutamate formed from DL-pipecolate-6-14C. The only previous evidence suggesting conversion of pipecolate t’o glutamat,e was the excret’ion of glutamat’e by cells oxidizing pipecolate (13). Since excreted glutamate might arise in ways only distantly relat,ed to pipecolate metabolism, we asked whet’her specifically labeled pipecolate formed labeled glutamate, and if so, whether the isotope w-as specifically or randomly incorporated. Glutamate isolat,ed from t,he supernat’ant liquid of cells oxidizing nn-pipecolate-6J4C was radioactive. Of 1.2 &i of the uL-pipecolate-6-14C added (sp. act. 429 dps/ctmole), 37 % was recovered as pipecolate and about 0.5 % each as glutamate (sp. act. 195 dps/pmole) and as a-aminoadipate. The decrease in specific activity was most likely due to dilution by endogenous glutamate. The isolated glutamate was next decarboxylated with hydrazoic acid and with chromic acids to liberate carbons 5 and 1 as COZ, respectively. The resulting COZ, 2,4diaminobutyrate and succinate were then isolated and counted (Table I). Degradation with hydrazoic acid liberated essentially all the radioactivity of glutamate as BaC03 from carbon 5. None was detected in t,he 2,4-diaminobutyrate from carbons 1 through 4. Although the results of chromic acid oxidation suggest that as much as 20% of the radioactivity of glubamate may reside in carbon 1, part of t,he succinic acid formed is further degraded to malonic and oxalic acids (21). We attribute the apparent discrepancy between t,he results of hydrazoic and chromic acid oxidation to liberation of CO2 from succinate. This view is consistent with the observed absence of detectable radioactivity in the 2,4-diaminobutyrate from carbons l-4 and its presence in the succinate from carbons 2-5. Carbon 6 of nn-pipecolate is thus converted to carbon 5

RODWELL TABLE

I~STRIBUTION FORMED

I

OF RADIO.XTIVITY IN GLUTAMATE FROM DL-PIPECOLATEBJYY Total radioactivity

Degradation procedure

Hydrazoic acid Hydraaoic acid Hydrazoic acid Chromic acid Chromic acid

Gilltamate degraded B&03

660 2125 3960 2940 2946

664 889 2105 222 236

(cpm)

Recovered as: 2,4-D&minobutyrate

s UCcinate

10 10 12 1054 855

a Twelve milligrams of nn-pipecolate-grown cells suspended in 1.2 ml ionic medium were preincubated at 30” for 30 min to reduce the level of endogenous pipecolate. One hundred rmoles (1.2 rCi) of nn-pipecolate-(i-i% were then added, and incubation was continued for an additional 45 min. Expired CO, was not collected. Cells were removed by centrifugation (3000 g; 5 min), washed with 1.0 ml of ionic medium, and the combined supernatant liquid and wash electrophoresed for 4 hr at 1500 V at pH 3.5. The glutamate, a-aminoadipate, and neutral amino acid areas were cut out, eluted with water, and counted. Electrophoresis at pH 6.5 and cellulose TLC in two solvents were used to confirm the identity and to determine the purity of the glut,amate and a-aminoadipate. Isolated glutamate was then degraded to determine the position of the isotopic label. Water degassed by boiling was used throughout.

of glutamate exclusively. Albhough the radioactivity recovered from glut’amate degradat,ions as BaC03, 2,4-diaminobutyrate, and succinate was considerably less than lOO%, when unreacted glutamate was taken into consideration recoveries of initial radioactivity ranged from 66 t’o 100% for both methods of degradation. Isotope from cr-aminoadipate-6-‘4, a known catabolite of pipecolate (13, IS), also was incorporated into glutamate by intact, pipecolate-grown cells. This was shown by long-term autoradiography of a pH 6.5 electrophoretogram of an incubation as described for pipecolate. While the experimental procedure was otherwise identical to that used above with pipecolate6-l%, insufFicient, radioactive glutamate was obtained to permit determination of the labeling pattern. Other products of pipecolate catabolism.

GLUTAMATE

FOI~MATIO~

Conversion of carbon 6 of pipecolat#e to carbon 5 of glutamate suggested participation of asymmetric intermediates. Possible candidates included a-aminoadipate, cyketoadipate, &aminovalerate, glutaryl-Cob, a-hydroxyglutarate, and cu-ketoglutarate. Washed pipecolate-grown cells incubated 8 min wi-ith nL-pipecolate-6J4C were extracted wi-ith aqueous ethanol, the extract, concentrated to dryness, dissolved in water, acidified, and continuously extracted with ether for 1s hr. Electrophoresis of the residual aqueous fraction at pH 3.5 followed bJ aut.ora~diography revealed ol-aminoadipate and glutamate, but no trace of &aminovalerate. The concentrated ether extract was fract,ionated by preparative cellulose TLC in benzene:ethanol:glacial acetic acid: : 45: 5: 8 (v/v). Autoradiography revealed dark bands at RF 0.0, 0.1-0.2, and 0.35-0.55 and two light bands of higher RF. After elution and rechromatography, the material at the origin was tentatively identified as isocitrate. Material in the RF 0.1-0.2 band was not identified. The RF 0.35-0.55 band was identified as succinate by t,wodimensional TLC both in propanol: eucalyptol: formic acid: : 5: 5: 2 (v/v) followed by xylene:phenol:formic acid: :5:5:2 (v/v), and in wamyl alcohol : formic acid : Hz0 : : 5: 1: 4 (v/v) (upper phase used) followed bs Ccl, containing 20 % glacial acetic acid. Sonic extracts of both pipecolate- and n&ate-grown cells were used to test conversion of possible intermediates to glutamate (Table II). Although bot,h extracts catalyzed glutamate formation, the activity of extracts of pipecolate-grown cells in all cases exceeded that of extracts of malategrown cells. Compounds are listed (Table 11) in increasing order of their efficiency of conversion to glutamate. Glutamate formation from n-aminoadipate and from (Yketoadipate was abolished and that from a-hydroxvglutarate was inhibited over 80 % by exclusion of OZ. Tra,samination of a-aminoadipate. Iteversible interconversion of a-aminoadipate and a-ket,oadipat e via t ransaminat ion was shown in extracts of pipecolate-grown cells. Incubat,ion mixtures contained, in 3.0 ml: 0.6 ml of ionic medium, 0.5 ml of dialyzed

FROM

PIPECOLATE TABLE

3,) II

Glutamate

formed

(nmoIes/mg/hr) by extracts of cells grown on: Substrate DLnkdate +oz

r)L-cY-Aminoadipate a-Ketoadipat,e oka-Hydroxyglutarate ol-Ketoglut.arate

10 10 32 580

DL-Pipecolate +02

18

22 55 805

-02

0.5
a Sonic extracts of cells grown to an equivalent bacterial densit,y on malate or pipecolate were dialyzed overnight against 0.1 M potassium phosphate (pH 7.0), assayed for protein, and diluted to 5.0 mg protein per ml. Aerobic incubations (+OJ were in test tubes and anaerobic incubations (-0,) in evacuated Thunberg tubes. Incubation mixtures contained, in 0.7 ml: SOpmoles potassium phosphate (pH 7.0), 25 pmoles L-aspartate, 2.0 mg of sonic extract protein, and 25 @moles of the indicated substrate. Incubation was for 1 hr at 30”. The glutamate present in 50.~1 aliquots was then isolat,ed by electrophoresis at pH 3.5 and quantitated as described under Methods.

supernatant liquid from a sonic extract centrifuged at, 105,OOOgfor 90 min of pipecolate-grown cells, and 12.5 pmoles each of an a-amino acid and an wketoacid. Combinations tested included waminoadipate plus pyruvate, oxalacetate or a-ketoglutarate, and cr-ketoadipate plus alanine, aspartate, or glutamate. After incubation for 2 hr at’ 30” the amino acids present were ident,ified by electrophoresis at pH 3.5 and by cellulose TLC in n-butanol: glacial acebic acid: HZO: : 12: 3: 5 (v/v). Incubation of a-aminoadipate with eit,her wketoglutarate, oxalacetate, or pyruvate under either aerobic or anaerobic conditions produced glutamate, alanine, and alanine, respectively. Conversion of oxalacetate to alanine rather than to aspartate reflects decarboxylation of oxalacetate to pyruvate before t ransamination. Aspartate can, however, act as amino donor. Incubation of a-ketoadipate with alanine, aspart ate, or glutamate produced a-aminoadipate. a-Ketoadipate (formed from cr-aminoadipate) and cu-ketoglutarate were identified by TLC of their 2,4-dinitrophenylhydrazones. An aliquot deprotcinized with tungstic acid

36

HARTLINE

AND

RODWELL

was treated with 2,4-dinitrophenylhydraamined for its ability to catabolize ar-hyzine reagent. Hydrazones isolated according droxyglutarate. Although incubation of the to Cavallini (22) were separated by cel- soluble fraction with a-hydroxyglutarate lulose TLC in n-but,anol:ethanol:2 M did not yield detectable products, the parNH40H: :70: 10: 20 (v/v) and by silica gel ticulate fraction readily catalyzed oxidation TLC in benzene: methanol: glacial acetic of a-hydroxyglutarate to a-ketoglutarate. acid: :45:5:8 (v/v). a-Ketoadipate 2,4- When aspartate was added traces of glutadinitrophenylhydrazone was identified by mate also were formed. Glut’amate formacoincidence of its mobility with that of a tion was substantially enhanced by the standard sample. Transaminase activity was addition of the soluble fraction which conpresent in t’he soluble, but not the particutains a-ketoglutarate:aspartate transamilabe, fraction of extracts centrifuged 90 min nase. Dehydrogenation of cr-hydroxyglutaat 105,OOOg and was unaffected by added rate to cr-ketoglutarate thus appears to be pyridoxal 5-phosphate or by the exclusion catalyzed by a particulate oxidoreductase. of O2 (Table III). This enzyme is the subject of a separate Since extracts of cells grow-n not only on paper (23). Induction of enzymes by growth on Lpipecolate but on malate catalyzed transamination of a-aminoadipate, we asked Zysine. Investigation of lysine-grown cells whether these extracts were equally active. was prompted both by the known metabolic Transamination studied in the direction of relationship between pipecolate and lysine ar-aminoadipate formation with aspartate in rats and by the low cost of lysine for and a-ketoadipate as cosubstrates was 3- to production of large quantities of cells. 4-fold faster in extracts of pipecolate-grown Lysine-grown cells were already known to be fully induced for L-pipecolate dehycells (Table III). Convemion of a-hydroxyglutarate to LY- drogenase (reaction 1, Fig. 1) and for L-W ketoglutarate and glutamate. Sonic extracts aminoadipate d-semialdehyde: NAD+ oxidoreadily catalyze glutamate formation from reductase (reaction 2, Fig. 1) (15, Meuth cr-hydroxyglutarate in the presence of as- and Rodwell, unpublished observations). partate and oxygen. This activity is more Experiments were conducted in a convenpronounced in extracts of pipecolate-grown tional manometric apparatus with cells grown either on L-lysine or on ammonium cells (Table II). A sonic extract of pipecolategrown cells was separated into soluble and DL-malate. No net oxidation of DL-a-aminoparticulate fractions by centrifugation at adipate, DL-pipecolate, or L-lysine was obcells. Using 105,OOOgfor 90 min and each fraction ex- served using malate-grown lysine-grown cells, net Qo, values of 485, 430, and 98 ~1 0, consumed/hr/mg dry TABLE III cells equivalent (13) were observed for L-ASPARTATEZff-KETOADIPATE AMINOTRANSFERlysine, pipecolate, and ar-aminoadipate, reASE ACTIVITY OF SONIC EXTRACTS~ -spectively. The Qo, for cr-aminoadipate is cY-Aminoadipate formed (nmoIes/mg/hr) by extracts of cells grown on: similar to that observed using pipecolategrown cells. Comparable results were obDL-Malate DL-Pipecolate +ot -0a +oa tained using sonic extracts. Utdization of m-pipecolai!e-6-14C and DIAY14 55 60 aminoadipate-6J4C. For a qualitative de13 40 52 termination of isomer utilization by intact a The sonic extracts and incubation conditions cells disappearance of DL-pipecolate-6-‘4C were those of Table II but using 25 @moles each of and DL-a-aminoadipate-6-14C from ethanolL-aspartate and of a-ketoadipate as substrates. soluble cell fractions was studied as a funcAerobic (+Oz) or anaerobic (-0%) incubation was tion of time (24). Pipecoiate-grown cells for 1 hr at 30”. The a-aminoadipate present in utilized both compounds at a first-order rate 50-~1 aliquots was then isolated by electrophoresis for over 90% disappearance of isotope from at pH 3.5 and quantitated as described under Methods. pipecolat,e and close to 50% disappearance

GLUTAMATE

FORMATION

FROM

PIPECOLATE

37

slow, radioactive glutamate would accumulat’e. In contrast, if entrance of a-aminoadipate into t,he cell was rate-limiting compared to the utilization of glutamate, only limit,ed amounts of radioactive glutamate may accumulate. The cu-aminoadipate excreted when cells are incubated with pipecolate was shown t,o be of the L-configuration DISCUSSION (13). It is conceivable that during incubaIn addition to the previously observed tion of cells with racemic cY-aminoadipat,e excretion of cu-aminoadipate-1-W by cells the n-isomer retards transport of the Loxidizing pipecolate-1-14C (13), in this work enantiomer.4 intact pipecolate-grown cells were shown to That cY-aminoadipate may be involved in excrete glutamate-5-14C when incubated the direct conversion of pipecolate to glutawith pipecolate-6-14C. The excretion of mate is suggested by the oxidative converasymmetrically labeled glutamate implies sion of a-aminoadipate, Lu-ketoadipate, athat glutamate is a direct metabolic product hydroxyglut)arate, and of a-ketoglutarate to of pipecolate and suggests that no symmetriglutamate. These oxidations occur in excal intermediates occur between pipecolate tracts of both malate- and pipecolate-grown and glutamate. SuccinateJ4C was identified cells, but are enhanced in all cases in piand isocitrateJ4C was tentatively identified pecolat,e-grown cells. These data suggest a as catabolites of pipecolate-6J4C degrada- sequence of induced enzymes for the contion in intact cells. Although the location of version of a-aminoadipate to glutamate. the radioisotopic carbons was not deterOther possible intermediates between (Ymined, we assume these to be t,ricarboxylic aminoadipat,e and glutamate are &aminoacid cycle intermediates resulting from valerate and glutaryl-CoA. No evidence was further degradation of glutamate. Metaboobtained for &aminovalerate formation from lism of succinate via the tricarboxylic acid a-aminoadipat,e using intact cells or cellcycle would yield cu-ketoglutarate-lJ4C free extracts of lysine-grown cells which with each revolution of the cycle. The cy- oxidize oc-aminoadipate and readily convert ketoglutarate-1-14C could in turn be transIysine t,o 6-aminovalerate.5 Conversion of aminated to glutamate-l-J4C. The absence a-ketoadipate to glutaryl-CoA has been of significant amounts of radioactivity in shown by Hayaishi et al. (23). Using comcarbon 1 of the isolated radioactive glutaparable conditions (26) we failed to detect mate may result from limited conversion of accumulat’ion of any product of cY-ketoadisuccinate to cr-ketoglutarate by the cells pate metabolism having an activated carunder the experimental conditions. boxy1 group. cu-Aminoadipate-6-14C also forms radioBoth a-aminoadipate (27) and cY-ketoactive glutamate but the locabion of the adipate (28) are excreted in the urine of isotope was not det,ermined. The limited rats and guinea pigs fed lysine and t,ransconversion of cu-aminoadipate-6-14C to radioactive glutamate, when compared with the 4 In a strain of Alcaligenes denitrificans isolated conversion of pipecolate-6-W to glutamate, by elect,ive culture on nL-a-aminoadipate as sole source of carbon and nitrogen and capable of does not favor cr-aminoadipate as an interof the Lmediate in the metabolism of pipecolate to growing on either isomer tttilization isomer is completely inhibit,ed until the n-isomer glutamate. However, the reduced accumulahas been metabolized (Hartline and Wood, untion of radioactive glutamate may result from differences in nn-pipecolate and DL-CX- published data). 5 Intact cells of a strain of Pseudomonas putida aminoadipate transport by the cells. If isolated by elective culture on DL-a-aminoadipat.e conversion of intracellular L-a-aminoadias sole source of carbon and nitrogen do not oxipate-6-14C (arising via pipecolate-6-14C) to dize &aminovalerate until a 45-min induction glutamat,e was rapid but the subsequent period has elasped (Hartline and Salvaterra, degradat,ion of glutamate \ras relatively unpublished data).

of nn-cu-aminoadipate-6-‘4C. Thus, both isomers of pipecolate and probably only one isomer of a-aminoadipate are utilized by pipecolate-grown cells. nn-a-Aminoadipategrown cells also use only 50 % of isotope from nn-a-aminoadipate-6-14C.

38

HARTLINE

AND

amination of a-aminoadipate occurs in mammalian liver (29). Transamination of cu-ketoadipate is also an obligatory step in lysine biosynthesis via a-aminoadipate (30) in yeast (31, 32) and Neurospora (31, 32). Pipecolate was previously shown to be converted to cr-aminoadipate by Pseudomonas putida (13, 16). That the next catabolic reaction may be transamination to a-ketoadipate is suggested by the enhanced ability of extracts of pipecolate-grown cells relative to malate-grown cells to catalyze transamination of a-aminoadipate. Pipecolategrown cells appear to be induced for higher levels either of a general transaminase or of a specific cr-aminoadipate transaminase. A specific a-aminoadipate transaminase functional for lysine biosynthesis occurs in yeast (33). Activity for a-hydroxyglutarate oxidation resides in the particulate fraction of cell-free extracts (22) while subsequent transamination of cr-ketoglutarate to glutamate occurs in the soluble fraction. This sequence of oxidation followed by transamination and the excretion of glutamate by intact cells leads us to suggest glutamate as a terminal catabolite of pipecolate degradation. The ability of lysine-grown cells, but not cells grown on malate, to oxidize lysine, pipecolate, and a-aminoadipate suggests that lysine induces synthesis of enzymes for catabolism of all three amino acids. Since comparable results were obtained with cellfree extracts, the inactivity of malate-grown cells is not due solely to failure of the amino acids tested to enter intact cells. Lysinegrown cells were shown to oxidize a-aminoadipate less readily than pipecolate. This may, in part, account for the previously observed excretion of ol-aminoadipate by intact cells oxidizing pipecolate (13). Previous investigations of pipecolate catabolism showed excreted (13) and enzymically formed a-aminoadipate (16) to be of the L-configuration. Resting cell suspensions of pipecolateor cY-aminoadipategrown cells utilized approximately half the initial amount of m-a-aminoadipate-6-W. Pseudomonas putida P2 thus may utilize only n-a-aminoadipate. Utilization of only half of the nn-a-aminoadipate even by DLa-aminoadipate-grown cells indicates that

RODWELL

catabolism of bhe n-isomer does not occur even when cells are grown in the presence of the n-isomer to permit induced utilization. Since pipecolate-grown cells use essentially all of the DL-pipecolate-6-14C, this organism can utilize both isomers of pipecolate. How n-pipecolate is catabolized is not kno1v-n. We consider it unlikely, however, that this proceeds via n-cr-aminoadipate. ACKNOWLEDGMENTS We thank Drs. David M. Greenberg and Morton Rothstein for gifts of nL-pipecolate-6-14C and of waminoadipate-6-i%, respectively. REFERENCES 1. ZACHARIUS, R. M., THOMPSON, J. F., AND STEWARD, F. C., J. Amer. Chem. Sot. 74, 2949 (1952). 2. HULME, A. C., AND ARTHINGTON, W., Nature London 170, 659 (1952). 3. MORRISON, R. I., Biochem. J. 63, 474 (1953). 4. LOWY, P., Arch. Biochem. Biophys. 47, 228 (1953) . 5. GROBBELAAR, N., AND STEWARD, F. C., J. Amer. Chem. Sot. 76, 4341 (1953). 6. SCHWEET, R. S., HOLDEN, J. T., AND LOWY, P. H., J. Biol. Chem. 211, 517 (1954). 7. ROTHSTEIN, M., AND MILLER, L. L., J. Amer. Chem. Sot. 76, 4371 (1953). 8. GROVE, J. A., GILBERTSON, T. J., HAMMER STEDT, R. H., AND HENDERSON, L. M., Biochim. Biophys. Acta l&l, 329 (1969). 9. HIGASHINO, K., AND LIEBERMAN, I., Biochim. Biophys. Acta 111, 346 (1965). 10. ROTHSTEIN, M., AND GREENBERG, D. M., J. Biol. Chem., 326, 714 (1960). 11. BORSOOK, H., DEASY, C. L., HAAGEN-SMIT, A. J., KEIGHLEY, G., AND LOWY, P. H., J. Biol. Chem. 176, 1383 (1948). 12. BAGINSKY, M. L., AND RODWELL, V. W., J. Bacterial. 92, 424 (1966). 13. RAO, D. R., AND RODWELL, V. W., J. Biol. Chem. 237, 2232 (1962). 14. BASSO, L. V., RAO, D. R., AND RODWELL, V. W., J. Biol. Chem. 237, 2239 (1962). 15. BAGINSKY, M. L., .~ND RODWELL, V. W., J. Bacterial. 94, 1034 (1967). 16. CBLVERT, A. F., AND RODWELL, V. W., J. Biol. Chem. 241, 409 (1966). 17. GIRI, K. V., RADH.IKRISHNAN, A. N., AND VAIDENATHAN, C. S., Anal. Chem. 24, 1677 (1952). 18. Kuo, M. H., SAUNDERS, P. P., AND BROQUIST, H. P., J. Biol. Chem. 239, 508 (1964). 19. GREENSTEIN, J. P., AND WINITZ,M., “Chemistry of the Amino Acids,” Vol. 3, p. 2547. J. Wiley New York (1961).

GLUTAMATE

20. MURPHY, J. B., 21.

22. 23. 24.

25.

26. 27. 28.

FORMATION

AND KIES, M. W., Biochim. Biophys. Acta 46, 382 (1960). WOLFE, H., in “Organic Reactions” (R. Adams, W. E. Bachman, J. R. Johnson, L. F. Fieser, and M. R. Snyder, eds.), Vol. III, p. 307. J. Wiley, New York (1946). CAVALLINI, II., A4~~ FRONTALI, N., Biochim. Biophys. Acfa 21, 270 (1954). KEITZ, M. S., AND RODWELL, V. W., J. Bacteriol. 100, 708 (1969). SMITH, IVORS, “Chromatographic and Electrophoretic Techniques.” Vol. I, p. 514. J. Wiley (Interscience), New York, (1960). HAYAISHI, O., NISHIZUKA, Y., ICHIYAMA, A., AND GHOLSON, R. K., J. Biol. Chem., 240, 733 (1964). HAYAISHI, O., NISHIZUKA, Y., AND KUNO, S., Biochim. Biophys. Acta 43, 367 (1960). BOULANGER, P., AND BISERTE, G., C. R. Acad. Sci. 232, 1451 (1951). CAVALLINI, I)., AND MONDOVI, B., Arch. Sci.

FROM

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39

Biol., Italy 36, 468 (1952). Chem. Abstr. 47, 3436 (1953). 29. BRAUNSTEIN, A. E., Advan. Protein Chem. 3, 1 (1947). 30. GREENBERG, I). M., AND ROD~ELL, V. W., “Metabolic Pathways” (D. M. Greenberg, ed.), Vol. 3, p. 341. Academic Press, New York (1969). H. P., AND STIFFEY, 9. V., Fed. 31. BROQUIST, Proc. 16, 198 (1959). 32. DEBOEVEE, T. G., “Enzymatic conversion of ketoadipic acid to aminoadipic acid in Fungi: Relation to lysine biosynthesis”, M.S. Thesis, University of Illinois, Urbana, Illinois, 1963. N., FJELLSTADT, T., AND OGUR, 33. PIEDISC~LZI, M., Biochim. Biophys. Res. Commun. 32, 380 (1968). 34. HARTLINE, R. A., AND ROD~ELL, V. W., Fed. Proc. 26, (1966).