Conversion of acetyl-coenzyme A into 3-hydroxy-3-methylglutaryl-coenzyme A in radish seedlings....

12
ELSEVIER Biochimica et Biophysics Acta 1211(1994) 85-96 BicxhimicaL et Bhphysiha &a Conversion of acetyl-coenzyme A into 3-hydroxy-3-methylglutaryl-coenzyme A in radish seedlings. Evidence of a single monomeric protein catalyzing a Fe”/quinone-stimulated double condensation reaction Thomas Weber a, Thomas J. Bach bp* a Botanisches Institut II, Universitiit Karhuhe, Kaiserstr. 12, D-76128 firlsruhe 1, Germany b C.N.RS.-I.B.M.P., D6partement d’En.zynwlogie Celkhire et Mokhlaire, Institut a’e Botanique, 28 rue Goethe, F-67083 StrasbourgCedex, France (Received 12 July 1993) Abstract We solubilized from radish membranes and purified to apparent homogeneity a monomeric protein (55.5 kDa) capable of catalyzing the two-step conversion of acetyl-CoA into 3-hydroxy-3-methylglutaryl(HMG~-CoA Unlike the situation described for other eukaryotes (yeast, animals), both enzyme activities needed for HMG-CoA synthesis (acetoacetyl-CoA thiolase, AACT and HMG-CoA synthase, HMGS) appear to be localized on a single polypeptide. Thus, the enzyme system is further referred to as AACI’/HMGS. The reaction as catalyzed by purified AACT/HMGS is strongly stimulated in vitro in presence of Fen-chelates (namely EDTA) and of quinone cofactors with pyrroloquinoline quinone (PQQ) being by far the most effective one studied so far. Whereas the Fen stimulation is apparently due to a V_ effect, PQQ increases the affinity of the enzyme system towards acetyl-CoA (1.9 pM vs. 5.9 PM, at 50 PM Fen, 100 pM EDTA, 20 PM PQQ). Stimulation by naphthoquinone (NQ) can be overcome in the presence of halogenated NQ-derivatives, while activation by PQQ remains unaffected, possibly indicating a much more specific-binding of the latter cofactor. Gel filtration experiments of enzyme after preincubation in presence of PQQ indicate that there is no covalent-binding of the quinone cofactor to the enzyme. As is also shown with partially purified enzyme from maize membranes, phenylhydrazine, known to react with PQQ as the prosthetic group of quinoproteins (see van der Meer et al. (1987) FEBS Lett. 221, 299-3041, efficiently inhibits the reaction. The data lead us to suggest a reaction mechanism that involves radical formation by the redox couple Fen/PQQ, thereby possibly facilitating the energetically unfavorable Claisen condensation as catalyzed during the first partial (AACT) reaction. Key words: Aldol condensation; Enzyme kinetic; Enzyme purification; HMG-Co& Isoprenoid biosynthesis; Radical formation; (Radish); (Maize) 1. Introduction HMG-CoA serves two functions: as the substrate for mevalonate biosynthesis, catalyzed by HMG-CoA re- ductase (HMGR, 3-hydroxy-3-methylglutaryl-coenzyme * Corresponding author. Abbreviations: AACT, acetoacetyl-coenzyme A thiolase; Br$ poly- oxyethylene ether W-l; IPTG, isopropyl-b-n-thiogalactoside; HMG- CoA, 3-hydroxy-3-methylglutarykoenayme A; HMGL, HMG-CoA lyase; HMGB, HMG-CoA reductase; HMGS, HMG-CoA synthase; NQ, 1,4_naphthoquinone; PQQ, pyrroloquinoline quinone; PVP, polyvinylpolypyrrolidone; SDS-PAGE, polyacrylamide gel eleo trophoresis in the presence of sodium dodecylsulfate. 0005-2760/94/$07.00 Q 1994 Elsevier Science B.V. All rights reserved SSDZ 0005-2760(93)E0219-A A reductase, mevalonate: NADP + oxidoreductase, CoA acylating, EC 1.1.1.34) and as a putative interme- diate in the degradation of branched-chain amino acids via HMG-CoA lyase (HMGL, (s)-3-hydroxy-3-methyl- glutaryl coenzyme A lyase, EC 4.1.3.4). Following the pioneering work of Harry Rudney and Feodor Lynen and their associates [l-5] for yeast and mammalian tissue, it has been well documented (see Ref. 6 and literature cited therein) that the conversion of three units of acetyl-CoA to HMG-CoA is catalyzed by two distinct enzymes: (a) acetoacetyl-CoA thiolase (AACT, acetyl-CoA acetyltransferase EC 2.1.3.9) catalyzing a Claisen type condensation and (b) 3-hydroxy-3-methyl- glutaryl-CoA synthase (HMGS, (S)-3-hydroxy-3-

Transcript of Conversion of acetyl-coenzyme A into 3-hydroxy-3-methylglutaryl-coenzyme A in radish seedlings....

ELSEVIER Biochimica et Biophysics Acta 1211(1994) 85-96

BicxhimicaL et Bhphysiha &a

Conversion of acetyl-coenzyme A into 3-hydroxy-3-methylglutaryl-coenzyme A in radish seedlings.

Evidence of a single monomeric protein catalyzing a Fe”/quinone-stimulated double condensation reaction

Thomas Weber a, Thomas J. Bach bp*

a Botanisches Institut II, Universitiit Karhuhe, Kaiserstr. 12, D-76128 firlsruhe 1, Germany b C.N.RS.-I.B.M.P., D6partement d’En.zynwlogie Celkhire et Mokhlaire, Institut a’e Botanique, 28 rue Goethe, F-67083 Strasbourg Cedex, France

(Received 12 July 1993)

Abstract

We solubilized from radish membranes and purified to apparent homogeneity a monomeric protein (55.5 kDa) capable of catalyzing the two-step conversion of acetyl-CoA into 3-hydroxy-3-methylglutaryl(HMG~-CoA Unlike the situation described for other eukaryotes (yeast, animals), both enzyme activities needed for HMG-CoA synthesis (acetoacetyl-CoA thiolase, AACT and HMG-CoA synthase, HMGS) appear to be localized on a single polypeptide. Thus, the enzyme system is further referred to as AACI’/HMGS. The reaction as catalyzed by purified AACT/HMGS is strongly stimulated in vitro in presence of Fen-chelates (namely EDTA) and of quinone cofactors with pyrroloquinoline quinone (PQQ) being by far the most effective one studied so far. Whereas the Fen stimulation is apparently due to a V_ effect, PQQ increases the affinity of the enzyme system towards acetyl-CoA (1.9 pM vs. 5.9 PM, at 50 PM Fen, 100 pM EDTA, 20 PM PQQ). Stimulation by naphthoquinone (NQ) can be overcome in the presence of halogenated NQ-derivatives, while activation by PQQ remains unaffected, possibly indicating a much more specific-binding of the latter cofactor. Gel filtration experiments of enzyme after preincubation in presence of PQQ indicate that there is no covalent-binding of the quinone cofactor to the enzyme. As is also shown with partially purified enzyme from maize membranes, phenylhydrazine, known to react with PQQ as the prosthetic group of quinoproteins (see van der Meer et al. (1987) FEBS Lett. 221, 299-3041, efficiently inhibits the reaction. The data lead us to suggest a reaction mechanism that involves radical formation by the redox couple Fen/PQQ, thereby possibly facilitating the energetically unfavorable Claisen condensation as catalyzed during the first partial (AACT) reaction.

Key words: Aldol condensation; Enzyme kinetic; Enzyme purification; HMG-Co& Isoprenoid biosynthesis; Radical formation; (Radish); (Maize)

1. Introduction

HMG-CoA serves two functions: as the substrate for mevalonate biosynthesis, catalyzed by HMG-CoA re- ductase (HMGR, 3-hydroxy-3-methylglutaryl-coenzyme

* Corresponding author. Abbreviations: AACT, acetoacetyl-coenzyme A thiolase; Br$ poly- oxyethylene ether W-l; IPTG, isopropyl-b-n-thiogalactoside; HMG- CoA, 3-hydroxy-3-methylglutarykoenayme A; HMGL, HMG-CoA lyase; HMGB, HMG-CoA reductase; HMGS, HMG-CoA synthase; NQ, 1,4_naphthoquinone; PQQ, pyrroloquinoline quinone; PVP, polyvinylpolypyrrolidone; SDS-PAGE, polyacrylamide gel eleo trophoresis in the presence of sodium dodecylsulfate.

0005-2760/94/$07.00 Q 1994 Elsevier Science B.V. All rights reserved SSDZ 0005-2760(93)E0219-A

A reductase, mevalonate: NADP + oxidoreductase,

CoA acylating, EC 1.1.1.34) and as a putative interme- diate in the degradation of branched-chain amino acids via HMG-CoA lyase (HMGL, (s)-3-hydroxy-3-methyl- glutaryl coenzyme A lyase, EC 4.1.3.4). Following the pioneering work of Harry Rudney and Feodor Lynen and their associates [l-5] for yeast and mammalian tissue, it has been well documented (see Ref. 6 and literature cited therein) that the conversion of three units of acetyl-CoA to HMG-CoA is catalyzed by two distinct enzymes: (a) acetoacetyl-CoA thiolase (AACT, acetyl-CoA acetyltransferase EC 2.1.3.9) catalyzing a Claisen type condensation and (b) 3-hydroxy-3-methyl- glutaryl-CoA synthase (HMGS, (S)-3-hydroxy-3-

86 T. Weber, T..i. Bach /Biochimica et Biophysics Acta 1211 (1994) 85-96

methylglutaryl-coenzyme A: acetoacetyl-coenzyme A lyase (CoA acylating), EC 4.1.3.51, catalyzing an aldol condensation. While the synthesis of HMG-CoA in mammals and yeast has been well examined and docu- mented, only little was known until recently as to the situation in plants [6-81. This article reports on the purification to apparent homogeneity and characteriza- tion of a membrane-associated enzyme from 4-day-old etiolated radish seedlings (&&anus sutivus L.), capa- ble of converting acetyl-CoA into HMG-CoA. This enzyme system, catalyzing both partial reactions, is further referred to as AACT/HMGS. Our observa- tions constitute evidence of plants having probably developed a further mechanism of HMG-CoA synthe- sis different from other eukaryotes.

2. Materials and methods

W-l) and incubated for 30 mm at 30°C. The resulting suspension was homogenized by 15 cycles with a Teflon homogenizer. After this treatment, enzymatic activity was found in the supernatant following a centrifugation at 100 000 X g for 45 min (Beckman L 2 65 B, rotor 60 Ti). Solubilized proteins in the supernatant were pre- cipitated with 2 vol. of -20°C acetone. After 3 min at 0°C the fluffy white precipitate was centrifuged (10 min, 2-4”C, 10 000 X g). The supernatant was immedi- ately decanted and the pellet was washed three times with buffer B additionally containing 0.3% Brij W-I (buffer 0. The washed sediment was homogenized and redissolved in 10 to 15 ml buffer D (like C, but contain- ing only 3 mM dithioerythritol [81X Insoluble proteins were removed by centrifugation (5 min, 5000 x g, 2- 4°C). Preparations thus obtained remained unstable and had to be further purified immediately by cohmm chromatography.

2.1. Materials 2.4. Column chromatography

The sources of chemicals and of radiochemicals All steps were performed with a HPLC/FPLC sys- have been described [8,91. tem (Pharmacia-LKB).

2.2. Isolation of membranes

Radish seedlings were grown in the dark and mem- branes (P 16000) were isolated as described [91. In brief, 300 g of intact seedlings were homogenized por- tionwise by the aid of a Waring blendor in ice-cold buffer A (200 mM K,PO, (pH 7.51, 350 mM sorbitol, 10 mM Na,EDTA, 5 mM MgCl,; before use this buffer system was supplied with 3 mM dithioerythritol and 5 g/100 ml insoluble polyvinylpolypyrrolidone) by three strokes of 5 s. The homogenate was squeezed through 15 layers of cheese cloth. The resulting ho- mogenate was then centrifuged for 10 min at 2000 X g to remove cell debris and PVP particles (Hermle cen- trifuge type ZK 400, rotor A 6.14, 4°C). The resulting supernatant was further subjected to centsifugation at 16 000 x g; the membrane pellet (P 16 000 X g), after careful removal of the floating lipid layer and of the supernatant, was resuspended in buffer B (50 mM K,PO,, 350 mM sorbitol, 10 mM Na,EDTA, 5 mM MgCl, and 10 mM dithioerythritol freshly added) and kept on ice until use. Maize seedlings were similarly grown on tap water for 7 days in complete darkness and membranes were prepared in the same way as described for radish.

An ion-exchange chromatography. The column (type 150/10, Merck, Darmstadt, volume 10 ml, filled w,ith Fractogel EMD TMAE 650, Merck) was firstly cleaned by passage of 10 ml 1.5 M KCl, followed- by equihbra- tion with > 60 ml of buffer D. After stabilization of the base-line of absorption (280 nm), up to 12 ml of the resuspended acetone precipitate were loaded at a flow rate of 0.5 ml/mm. After 24 min (end of loading phase) the flow rate was increased to 1.5 ml/r& and the column was washed for an additional 20 min with buffer system D. Proteins were eluted by a gradient of 0 to 1 M KC1 in buffer D over 30 mm.

Dye-ligand chromatography. Tk same type of col- umn was used, filled with Fractogel TSK AF-Grange (Merck) and the material was equilibrated with buffer D to which 15% (v/v) 150 mM K,PO, -at pf-l 7.5 was added (buffer E). After base-line stabilization, the’mosf active pooled fractions from the ion-exchange chro- matography (10 ml) were loaded at a flow rate of 0.5 ml/min. After the end of the loading phase (24 min), the flow rate was increased to 1.5 ml/min and- the column was washed with modified buffer D for another 10 min. Proteins bound to the column were eluted. over 35 min by the aid of a linear gradient of’ modified buffer D to buffer A, additionally containing 3 mM dithioerythritol, 0.3% Brij W-l and 600 mM KC1 (buffer F).

2.3. Enzyme solubilization

For the purification of AACT/HMGS, proteins were solubilized from membranes by the aid of Brij W-l [6]. 2 Vol. of suspended membranes were mixed with 1 vol. of 6% (w/v> polyoxyethylene ether (Brij

Gel filtration. A column type 16/70 (Pharmacia, volume 110 ml, filied with Superose 6 prep. grade, max. loading volume 10 ml) or type 600/10 (Merck, volume 40 ml, Fractogel TSK HW 50; max. loading volume 6. ml) were used for molecular sieving. Columns were washed with two void volumes of buffer B. After stabi-

T. Weber, TJ. Bach /Biochimicu et Biophyska Acta 1211 (1994) 85-96 87

lization of the base line at 280 nm, the two most active fractions from the affinity chromatography were pooled (usually 5.5 ml) and loaded at a flow rate of 0.75 ml/mm. The column was eluted under the same con- ditions. The molecular mass was determined by com- parison to appropriate molecular weight standards.

Purification of HMG-CoA reductase. HMGR from yeast was purified according to Kirtley and Rudney [lo] with little modification (i.e., addition of 5 mM dithio- erythritol to all buffer solutions) except the omission of the Zn-acetate precipitation step. As a final step, affin- ity chromatography on HMG-CoA agarose was added. As an enzyme source, we also used E. coli overexpress- ing the soluble 45 kDa domain of radish HMGRl [HI. 1 1 of cells were grown at room temperature until the absorbancy was > 0.6, followed by induction with IPTG. Cells were harvested after a further 24 h and homogenized by ultrasonication. From the homogenate cell debris were removed by a centrifugation at 10000 x g (10 min, 4°C). HMGR was purified from a 100 000 x g supematant by a 50% ammonium sulfate precipi- tation. The precipitate was redissolved in buffer D and was stable at -20°C for several weeks. This step was followed by affinity chromatography on 11 ml Fractogel TSK AF-Blue (Merck) with a gradient of buffer D to buffer D plus 3 M KCI. HMGR eluted at about 1.5 M KCl. Upon SDS-PAGE analysis only the 45 kDa band was detected. Final specific activity was approx. 5 pmol min-lmg-l.

the samples were heated to 110°C for 3 h. Under these conditions, thioesters are hydrolyzed; only enzymati- tally formed i4C-HMG acid remains in the vials, whereas [ 14C]acetate is volatile. To redissolve the HMG acid, 0.25 ml of water was added followed by incuba- tion at R, for 10 min. After addition of 4 ml scintilla- tion cocktail (Aquasafe, Zinsser, Frankfurt) the sam- ples were vigorously vortexed. After 10 min, radioactiv- ity was determined by the aid of a liquid scintillation counter type 2000 CA (Packard) with automatic quench correction by the external standard method. Appropri- ate blinds were run in the absence of enzyme protein. Data were calculated and plotted as described [8,9].

HMG-CoA lyase. This enzyme was assayed as de- scribed [6,8]. Briefly, 25 ~1 enzyme solution were added to 15 ~1 of a mix containing 14 ~1 200 mM Tris-HCl (pH 8) and 1 ,ul 1 M MgCI, in H,O. The reaction was started by addition of 10 ~1 of a solution consisting of 2 yl (RS)-[3-14ClHMG-CoA in 50 mM K,PO,, pH 4.5 (ca 40000 dpm, 8 PM final concentration in the assay; only 50% of this synthetic substrate can be used up by HIMGL, due to its exclusive stereospecificity towards (S)-HMG-CoA) and of 8 ~1200 mM Tris-HCI (pH 8). After up to 20 min of incubation the reaction was stopped by addition of 125 ~16 N HCI and the samples were further processed as described for the assay of AACT/ HMGS activity.

HMG-CoA reductase. Enzyme activity was assayed radiochemically as described in detail [9].

2.5. Enzyme assays 2.6. Protein determination

Radiochemical assay of AACT/HMGS activity. The assay for AACI’/HMGS is based on the method de- scribed by Clinkenbeard et al. [12] with some modifica- tions [8] and further optimizations; where not further indicated (standard conditions), the enzyme was incu- bated for 5 min in the presence of 100 mM Tris-HCI (pH 7.5),20 PM [l- “C]acetyl-CoA (s.a. 13 &i/pmol), 0.5 mM Na,EDTA, 0.25 mM FeSO,, 10 ,LLM PQQ, 300 ng of purified AACT/HMGS or up to 300 pg of crude protein in 20 ~1 buffer D in a total assay volume of 50 ~1. The enzyme solution (20 ~1) was mixed with a freshly prepared solution of 17 ~1 200 mM Tris-HCl (pH 7.51, 1 ~1 25 mM Na,EDTA (pH 7.01, 1 ~1 12.5 mM FeSO, (pH 2.01, 1 ~1 0.5 mM PQQ in H,O (pH 7). The reaction was started after 3 min of preincuba- tion at 30°C by addition of 10 ~1 substrate mix (1.25 ~1, 0.2 mM [1-‘4C]acetyl-CoA in 50 mM K,PO, (pH 4.51, approx. 30000 dpm, 0.4 ~1 unlabeled acetyl-CoA (2 mM), 1.9 mM in 50 mM K,PO,, 8.35 ~1 200 mM Tris-HCl (pH 7.5). After 5 min, the reaction was termi- nated by addition of 125 ~1 6 N HCl. In kinetic experiments, the incubation was shortened to 2 min, and substrate and cofactor concentrations were varied as appropriate. After transfer to scintillation minivials,

Protein was determined by a Lowry method [13] with some modifications [9].

2. Z SDS-polyacrylamide electrophoresk

Vertical SDS-PAGE was by the method of G&g et al. 1141 using a Protean II slab cell (Bio-Rad) and 10% gels. Silver staining was according to Heukeshoven and Demick [ 151.

2.8. Recrystallization of HMG acid

A large-scale incubation of [14C]acetyl-CoA (250 ~1 volume, total radioacitivity 250000 dpm, 30 min at 3O”C, in the presence of highly purified AACT/ HMGS) was processed as described above until the 3 h heating step for the standard test system. Unlabeled HMG-acid (200 mg, Fluka) was added, followed by 0.5 ml EtOH. The sample was transferred to a pre- weighted glass vessel and the solvent was evaporated under vacuum. After withdrawal of an aliquot (de- termination of initial radioactivity), crystallization cy- cles from diethyl ether (dried over KOH) were re-

88 T. Weber, TY. Bach /Biochimica et Biophysics Acta 1211 (1994) 8.5-95

peated until no further change in specific radioactivity could be observed.

3. Results

3.1. Enzyme purification

We developed a purification protocol for the mem- brane-associated enzyme system AACT/ HMGS, which leads to the apparent homogeneity. The purification fold is 731, with a specific activity of the protein of 181.9 mnol mg-l min-’ (standard assay conditions) (Table 1).

The main steps of this purification protocol consist of (a) acetone precipitation and (b) dye&and affinity chromatography. The enzyme was very sensitive to high salt concentrations, rendering ammonium salt precipi- tation impractible. Among a number of methods tested (see Refs. 6,8,16), the introduction of a precipitation step with cold acetone proved the most effective for concentrating the enzyme without a complete or nearly complete loss in activity. Anion-exchange chromatog- raphy on Fractogel EMD TMAE 650 (where AACT/ HMGS does not bind) reliably removed HMGL, which might interfere with AACT/HMGS activity. The most important step to achieve a considerable purification factor was a rather selective dye-affinity material AF Orange (Merck), where the enzyme system binds and is eluted at moderate salt concentrations without appre- ciable loss in activity. The final gel filtration step, which removed low molecular weight contaminants, led to an activation of AACT/HMGS due to the removal of excess KC1 introduced during the elution from the affinity chromatography.

SDS-PAGE of the purified enzyme followed by sil- ver staining showed a single protein band with an apparent molecular mass of 55.5 f 2 kDa (Fig. I>. This value is similar to that determined by gel filtration on various materials under non-denaturing conditions [8], thus indicating that the active protein apparently is monomeric.

3.2. Validity of the assay method

We have shown earlier, even with rather crude AACT/ HMGS preparations, that the i4C-labeled product of incubations with [l-i4C]acetyl-CoA, in the presence of HMGR and NADPH, could- be quantita- tively converted to MVA [6]. By using highly purifed AACT/HMGS we can show that the presence of an excess amount of HMGR even leads to a higher appar- ent activity of AACT/HMGS, possibly due to the removal of the immediate enzymatic product (Table 2). This effect, expressed in percentage of IIMG-CoA formation, is especially drastic in those cases where the activating cofactors Fen and PQQ were absent (see below), resulting in a low synthesis of I-IMGCoA; Recrystallization of enzymatically formed ‘“C-labeled product diluted with unlabeled HMG acid led to > 90% apparent radiochemica1 purity, a value that matches the purity of the substrate as indicated by the manufacturer.

3.3. Cofactor requirements

Studies on the requirement of cofactors, e.g., to overcome disactivation effects during the development of purification protocols (see Ref. 6), revealed that for maximum in vitro activity the enzyme needs chelated Fen [8]. A series of bivalent cations at constant concen- trations of 250 PM were tested for their ability- to activate completely purified AACT/HMGS in vitro using an enzyme from three different purifications (Fig. 2). Fen was by far the most active cation and- some activation could only be achieved in the presence of such cations that could be oxidized, -e.g.,.Mn? In an extension of an earlier study with semi-purified AACT/ HMGS [8], various chelators were examined as to whether they amplified or blocked the enzyme acti- vation by Fen (data not shown). The results were essentially the same as observed with partially &XT/ HMGS. As was shown earlier, EDTA was most active, followed by citrate, the latter known to be a natural chelator of Fenin the living cell [17]. The data indicate

Table 1 Solubilization and purification of AACT/HMGS from 4-day-old etiolated radish seedlings The protein has been solubilixed and purified as described in Section 2

Volume Specific activity Purification (ml) (nmd mg-’ min-r) factor

P16OOOxg 37 0.25 1 P160OOXg+Brij 52 0.78 3.1 Acetone precipitate 35 0.45 1.8 Anion-exchange 36 4.2 16.7 AP Orange 24 41.0 165 Gel filtration 24 181.0 731

Yield Protein content (o/o) OngI

100 677 173 374

7 294 86 35 25 1.0 63 0.6

AA

T. We&, T.J. Bach /Biochimica et Biophysics Acta 1211 (1994) 8.5-96 89

A B C D

Fig. 1. Silver-stained SDS-gel of purified radish AACT/HMGS. Lane A, 1.5 ~1 of fraction 5 from anion-exchange column; lane B, 5 ~1 of fraction 19 from dye ligand chromatography; lane C, 20 ~1 of fraction 5 from gel filtration (without detectable activity, control); lane D, 20 ~1 of fraction 7 from gel filtration, concentrated S-fold by freeze dry& at the right: molecular weight standards (1) myosin, (2) ~-gala~o~d~, (c) phosphoryIase b, (d) bovine serum albumin, (5) ovalbumin, (6) carboanhydrase). The gel had been heavily overstained to detect any impurities present. The location of AACI/HMGS is indicated like that of the large subunit of ribulosebiiphosphate carboxylase (Rubiico), which was independently identified by Western blot analysis using spinach protein extracts and antibodies kindly provided by Dr. Radunz, Rielefeld,

n no PPP

g 500-- IlopMPpPa c s E 400--

Concentration Ion/EDTA 250/500 pM

g B 300-- ‘5

F zoo-- 1. I

Contr. Mn2+ Co2+ Sn2+ Ni2$’ Fe3+ Fe2+

Fig. 2. Effect of various ~TA~ela~d cations, on the in vitro activity of highly purified radish AACI/HMGS. The enzyme prepa- ration was further subjected to gel filtration on a PD 10 column in presence of buffer D (containing only 1 mM dithioerythritol) in order to remove low molecular weight components.

a correlation between the degree of activation and the stability of the complexes. Fen complexed with CN-, where all six coordination sites are occupied, was inac- tive. Chelators such as EDTA protect Fe” from oxida- tion and p~ticul~ly under slightly basic unctions 1181. Furthermore, under our assay conditions, viz. in the presence of about 3 mM dithioerythritol, Fen’ is reduced rather rapidly (data not shown).

Since ~hibitio~ studies in the presence of hydroxy- urea, a quencher of radicals [191, indicated the forma- tion of radicals being involved in the in vitro stimula- tion of AACT/kIMGS activity [81, we examined the effect of various quinones known to stabilize unpaired electrons. Indeed, in the presence of quinones, the activation by Fe” could be further increased, with PQQ having the highest efficiency of all compounds

Table 2 Conversion of en~ti~~ formed HMG-CoA into mevalonate in the presence of radish HMGRl overexpressed in and purified from E. coli

Condition Product of AACT/HMGS reaction converted into mevalonate (in dpm and percentage of HMG-CoA formed in the absence of HMGR and NADPH) B

Ekpt.1” Expt. II Expt. III Average

Without Fe’r/EDTA/PQQ 3 347 (224%) 3 450 (246%) 1169 (202%) 224% 100/200 pM Fe”/EDTA 6 583 (110%) 6 629 (110%) 2971(142%) 121% 100/200/10 PM Ferr/EDTA/PQQ 16 256 (119%) 20448 (149%) 9 545 (115%) 126% Dito, + 1 mM DTE 19996 (142%) 20454 (145%) 11046 (106%) 131%

a R~iola~led product of fl-“C&cetyl-CoA conversion in presence of purified radish AAcT/HMGS when assayed under the conditions as indicated. HMGR activity itself is not stimulated or inhibited in presence of Fen or PQQ. b The experiments have been conducted by using three different enzyme preparations.

90 i? We&r, T.J. Bach /Biochimica et L3iophysica Acta 1211 (1994) 85-96

_-_ U no Fe’+/EDTA a-l ,4 Naphthoquinone

a ?OO m 50/l 00 jbM Ftv’+/EDTA

_ Contr. 25 50 100 200 400

Nophthoquinon~ jjtFA]

a 7oo I 0 no Fe’+/EDTA

iSI9 50/1OD PM Fe2+/EDTA

Vitamin K1

; 600 - lOO/‘ZOO @M Fe2+/EDTA

z 500

100

D Contr. 25 50 100 200 500

Vitamin Kl [/IM]

600 f

U no Fe2*/EDTA Men~dione s z 700-- L 5l 50/l 00 ,uM Fe2+/EDTA

I

Contr. 25 50 100 200 530 ~~adione blk(]

ESY 50/100 @4 Fe

Contr. 1 5 10 15 25

PM @l]

OH-DOPA 7oo

6oo

t

C3 no Fe’+/EDTA

ES3 SO/‘100 J&+ Fe2+/EDTA

I 100/200 @.I Fe2+/EDTA

500 = 50/100 rM Fe’+/EDTA,

400 t

(Ok-DO+A oxidizeb by H202)

Conk. 5 10 15 25 50

OH-DOPA [jrM]

Fig. 3. inundation-dependent st~ula~ion of in vitro activity of purified radish AACT/HMGS by various quinones in the absence or presence of Fe”/EDTA. Activities are expressed as percent of control. The activation by Fe II alone was not considered, because onty the relative effect of quinones was to be shown. At SO/100 PM Fen/EDTA, AACT/HMGS activity is stimulated by about ZOO%, at 100/2OO bM by about 300%. Thus, an activation by 600% (e.g., in Fig. Sa, last column) would translate into an ll-fold increase in apparent activity over the control measured in the complete absence of exogenous cofactors.

tested so far (Fig. 3). The rest&s presented here show that this quinone cofactor must be rather hydrophilic than lipophilic because the activation effect is clearly correlated with hydrophilici~ (see the series naphtho- quinone - menaquinone - phylloqu~o~e = vitamin K,). Hydroxy-DOPA, also described as a possible co- factor of quinoproteins [20] is much less active than PQQ, although it shares some structural features with

the Iatter compound. When oxidized by the aid of hydrogen peroxide, its activity is increased, but still .well below that of PQQ.

That PQQ has a high affinity towards AACT/ HMGS is further indicated by the observation that within a series of 3-halogenated derivatives of I$- naphthoquinone carrying additional 2-methyl- or 2-pro-- pyl groups, originally designed as putative inhibitors of

T. Weber, TJ. Bach /Biochimica et Biophysics Acta 1211 (1994) 8.5-96 91

0 250/500 pM Fe2+/mrk A = 114 NQ no POP In the assay 6 = 3-Cl-2-CH3-NCl

C = 3-Cl-2-C3H7-NQ

D = 3-Br-2-CH3-NP

E = 3-Br-2-C3H,-NQ

cont. A C D E

Concentration of each chemical is 100 PM

Fig. 4. Effect of halogenated l+naphthoquinone (NQ) derivatives on the NQ- and PQQ-mediated stimulation of purified radish AACT/HMGS. The inhibitors were added in lo-fold excess as compared to the concentration of PQQ. Control: measured in the absence of PQQ, but in presence of 250/500 PM Fe=/EDTA. White cohmms: in the absence of PQQ. In samples B,C,D,E (white columns) besides 100 PM of the halogenated derivatives, 50 pM NQ. Black columns indicate the presence of 10 PM PQQ in the assay mixture. Bars indicate the mean of three independent experi- ments (three different preparations of purified AACI’/HMGS).

photosystem II activity [211, these compounds can effi- cieu’ly block enzyme activation by naphthoquinone, but not by PQQ (Fig. 4). Even a 2-fold increase in concentration of naphthoquinone derivative is suffi- cient to prevent naphthoquinone-induced activation.

Preincubation of purified AACT/HMGS at 20°C in the presence of 10 PM PQQ and at varying concentra- tions of Fe”/EDTA led to some time-dependent in- crease of apparent activity (Fig. 5). To further eluci- date the mechanisms of interaction with Fen and PQQ, we incubated aliquots of purified AACT/HMGS in the presence of 50 ,uM Fe”/100 ,xM EDTA, or with 25 PM PQQ and Fe”/EDTA (control: complete ab- sence of cofactors). Aliquots of 1 ml were subjected to

z 300 0 no Fe2+,‘EDTA

$ III 50/l 00 fl F~J~+/EDTA

g h19 100/200 /JM Fe2+/EDTA 0 240

%

t

I 250/500 /dd Fe2+/EDTA

j&. 160

b :z 120

ti

60

n _ . 1 2 3 5 10

Pt-aincubation (1OpM PM) [min]

Fig. 5. Time course of PQQ-induced activation of AACT/HMGS. During preincubation in the presence of PQQ (10 &f) and of increasing concentrations of Fen/EDTA the samples were kept at 20°C. 100% (control> corresponds to enzyme tests without preincuba- tion.

Table 3 Gel filtration of purified radish AACT/HMGS after preincubation in the presence of Fe”/EDTA and/or of Fe’/EDTA plus PQQ

Expt./ Activity (expressed in dpm) measured in presence of Fraction a [r?lAc- [‘4CL40 [14C]Ac-CoA

COA CoA+Fen + Fen + PQQ

I/3 115 163 779

I/4 762 2160 19451

I/5 396 1210 16401 II/3 102 115 409 II/4 663 1845 15 372 II/5 556 1485 19909 III/3 103 109 274 III/4 650 2120 14729 III/S 500 1688 19112

a (I) Radish AACT/HMGS (1 ml, purified as described in Table 1) was subjected to gel filtration on a PD-10 column (control). (II) The enzyme was preincubated in the presence of SO/l00 I.IM Fe”/EDTA and then put onto the column. (III) The enzyme was preincubated as under II), but further in the presence of 25 PM PQQ. 12 fractions were collected from each column stepwise eluted by a phosphate buffer system (containing dithioetythritol, needed to keep the en- zyme active, plus 0.3% (w/v> Brij W-l). Each fraction was then assayed under the following condition: (a) Only in the presence of [14C]acetyl Co& (b) [14C]acetyl-CoA plus SO/l00 PM Fen/EDT& (c) like under (b), but in the presence of 25 PM PQQ. From 12 fractions collected each only the active ones are shown.

gel filtration on PD-10 column. l-ml-Fractions were collected and enzyme activity was determined in the absence or in the presence of Fe”/EDTA and of PQQ. Evidently, binding is not strong enough that a passage through a PD 10 column after 10 min of preincubation could not largely remove the quinone (Table 3). However, AACT/HMGS activity can be fully restored after readdition of PQQ and Fen. It is thus unlikely that the quinone cofactor is covalently bound to the protein. There is a little shift in the elution pattern, because AACT/ HMGS-PQQ is slightly retained on the column as compared to enzyme that was not preincubated in presence of Fen/PQQ, indicating some interaction with the Sephadex mate- rial. Furthermore, PQQ might be specifically adsorbed by Sephadex, thereby facilitating the dissociation.

3.4. Kinetic properties

While the presence of chelated Fen only increases V_ (Fig. 6a), PQQ increases V_ and the affinity of the enzyme system towards its substrate acetyl-CoA by lowering the K, value from 5 to 1.9 PM (Fig. 6b). Evidently, both cofactors co-operate quite closely in the apparent activation of AACT/HMGS. Without Fe”/EDTA in the assay, the activation of AACI’/ HMGS by PQQ is quite negligible; this residual value can be readily explained by the ubiquitous presence of trace amounts of Fe in most chemicals commercially available. (we calculated a minimum concentration of

92 T. Weber, T.J. Bach /Biochimica et Biophysics Acta 1211 (1994) 85-96

0.12 0.72

a.3 0 l - l 1 O/20 fl Fe/EDTA A-A 25/50 @.i FefEfIlA

O.‘O

. - A 50/l 00 pM Fe/EDTA 0.06 II - n 1 DD/2W pM Fe/ElJTA 0.08

PPP in all aasqs: 0.06 0.06

0.04 0.04

0.02 0.02

0.00 0.00 -0.40 -0.30 -0.20 -0.10 0.00 0.10 0.20 0.30

l/S IirM ocatyl-caA]-’

0.10 I rO.10

i:

p 0.06 --

f 3 0.06~-

Y 4 0.04”.

5

: 0.02 -.

:

o-0noPQQ 0-0 2puPpPP .%-A 5@PQ9 A--* 1OpMPQP n-n m/&M Pcta

0.06

0.06

Fe*+‘DTA in all way% 50/100 pM 0.04

0.02

0.00 0.00 0.20 0.40

l/S bM acetyl-CoA]-’

Fig. 6. Double-reciprocal plots for the determination of kinetic constants of purified AACT/HMGS in the presence of varying concentrations of substrate and cofactors. (AI At 5 PM PQQ and increasing concentrations of Fer’/EDTA and fB> at a constant inundation of SO/l00 ,uM Fen/EDTA and increasing concentra- tions of PQQ. Incubation time in all tests was 2 min in order to exclude enzyme denaturation and to keep substrate consumption below 30%.

about 10 @M Fe in our enzyme assay mixture.> Free HS-CoA and some selected CoA esters were only moderately inhibitory to AACT/HMGS (data not shown). This holds true for immediate products of the reaction, HMG-CoA and HS-CoA, but at 100 PM acetoacetyl-CoA inhibits the enzyme by about 50%.

Table 4 Activation of partially purified maize AACT/HMGS by Feu/EDTA and PQQ (mean of three independent experiments with different enzyme preparations+S.D.I

JZxperiment Activiw in % of control

Without Fe”/EDTA/PQQ (control) 100 With Fe”/EDTA (250/500 PM) 288 f 12.6 With Fe”/EDTA/PQQ (250/500/10 PM) 430f24.8

Maize AACX/HMGS was partially purified via detergent-solubiliza- tion, acetone precipitation and anion-exchange chromatography. Be- fore use in these experiments, enzyme solutions were further sub- jected to a gel fihration on a PD 10 cohmm equilibrated with a buffer system containing 50 mM I&PO, pH 7.5,350 mM sorbitol, 10 mM NarEDTA, 5 mM MgClz, 1 mM DTE, 0.3% Srij W-l. Sub- strate concentration in all assays was 10 PM.

100 a-Ahc.thnez2mln 1

0 ,.-Olne.tim5min

35 45 55

Temper&we foe]

.

e .

\

\

1 3.0 3.1 3.2 3.3 3.4 3.5 3.6

1000/i [K-l]

Fig. 7. Temperature optimum of purified radish AACI’J/HMGS. Incubations were done for 2,s and 10 min. Approx. Va values were determined by extrapolation using spline functions toeelculate best fit curves [45f The slope as determined from the linear part of the corresponding Arrhenius plot yields a value of 75.8 W)mol -as

apparent activation energy.

The enzyme assay conditions, under which maximally 30% of substrate were converted to- product, were designed so that an inhibitory concentration of.tiroduct was never reached. Thus, such a potential, effect was neglected when kinetic constants were calculated. The

Table 5 Inhibition by phenylhydr~in of partially purified maize AACT,# HMGS

[Phenylhydrazin] Without Fe”/ With Fe”/ EDTA/PQQ EDTA/PQQ

10 PM 48.7*4.1 74.6+5.4 loo J&M 22.3 f 4.0 42.2 f.4.4 1mM 14.4*4.5 29.5 L 4.8 2mM 12.4 f 4.2 25.7f5.0

Activity is expressed in percent of control, determined in the absence of the inhibitor; mean of three independent experiments with differ- ent enzyme preparations f SD. Maize AACT/HMGS was partially purified via detergent-~olubiliza- tion, acetone precipitation and ~ion~~~ge c~ato~aphy. Be- fore use in these experiments, enzyme solutions were further sub- jected to a gel filtration on a PD 10 column in 50 mM K,PO., tpH 7.5) in order to remove sorbitol that.also reacts with‘phenylhydra- zinc. Substrate concentration in all assays was 10 PM.

T. Weber, T.J. Bach /Biochimica et Biophysics Acta 1211 (1994) 85-96 93

e

t

m no Fe2+/EDTA/PCtP: Maize

z 160 KY 250/500/20 pM Fe2+/EDTA/PQCk Maize

! I no Fs2+/EDTA/PQQ: Radish

7s 120 0 250/500/20 pM Fe2+/WMjPQp: Radish

10 100 1000

Phenylhydrazine by]

Fig. 8. Effect of phenylhydraxine on the activity of highly purified radish AACT/HMGS and semi-purified maize AACT/HMGS. The radish enzyme was purified as indicated in Table 1. The maize enzyme, solubilixed from a P 16000X g isolated from 7-day-old etiolated seedlings, was purified via the anion-exchange step. In order to remove the sorbitol, in which unavoidable impurities will also lead to a reaction with/consumption of phenylhydraxine, the buffer system was exchanged by a passage of 2 ml enxyme solution through a PD 10 column equilibrated with 50 mM K,PO, pH 7.5 (final volume: 3.5 ml). [l-r4C]acetyl-CoA concentration in the assays was 10 PM. Protein solutions were preincubated in the presence of phenylhydraxine for 5 min at 30°C before addition of substrate mix.

pH optimum was around 8.0 (not shown). For the sake of substrate and cofactor stability, under standard con- ditions a pH of 7.5 was used where enzyme activity exhibited about 95% of maximum activity. The activa- tion energy extrapolated to V, conditions was 75.8 kJ/mol. The enzyme had a maximum activity at an incubation temperature of 35”C, which was shifted to 30°C when it was incubated longer than 2 min, indicat- ing denaturation to commence above 30°C (Fig. 7a,b).

That we have probably identified a new quinopro- tein-type enzyme is supported by the observation (Fig. 8) that its activity can be blocked in the presence of phenylhydrazine, known to specifically interact with ketone groups of (protein-associated) quinone [22]. Al- though it was not yet possible to use the same protocol of purification for a solubilized enzyme preparation from maize, in vitro activation by Fe”/PQQ is clearly visible, even though less prominent at this stage of purification (Table 4). Inhibition of semi-purified maize enzyme by the phenylhydrazine (Table 5) provides fur- ther evidence that plants in general might have devel- oped a pathway of HMG-CoA synthesis that differs from that of animals.

4. Discussion

In our initial attempts to characterize HMGS alone we used an assay adopted from Clinkenbeard et al. [12], with 14C-labeled acetyl-CoA and unlabeled ace- toacetyl-CoA in the assay mixture. Only later did we

find that the enzyme system AACT/HMGS did not need acetoacetyl-CoA to catalyze the formation of HMG-CoA [16]. Optical assay methods for AACT and HMGS exist [23], but especially with crude prepara- tions of plant proteins proved to be very unreliable. During the establishment of purification protocols, we had always assayed fractions twice: (a) in the presence and (b) in the absence of acetoacetyl-CoA. If a protein fraction exclusively contains HMGS but not AACI activity, should we have been able to observe the incorporation of radiolabel into HMG-CoA only when both substrates were available. However, with none of the various methods tested could we achieve a separa- tion of AACT from HMGS. Thus we speculated that both enzymes might form a very close complex that cannot be separated [6,8]. However, the relatively low apparent molecular mass (about 56 kDa) of radish AACT/HMGS as determined by non-denaturing gel filtration on various materials was somewhat puzzling and seemed to contradict the hypothesis of having an enzyme complex catalyzing the double condensation.

Our earlier observation that it is impossible to trap any intermediate acetoacetyl-CoA in the assay mixture in presence of mammalian /3-hydroxybutyryKoA de- hydrogenase and of NADH [6,16], indicating that there is no release of this putative intermediate into the surrounding medium, is now further substantiated by our finding of a single protein apparently catalyzing both reactions. Further evidence is provided by the fact that the antibiotic F-244 [24,25], which efficiently in- hibits yeast and mammalian HMGS, is completely inef- fective in our system [26], probably because the puta- tive site of interaction, if existing at all, is somehow shielded in the radish AACT/HMGS. Acetoacetyl- CoA itself is a rather potent in vitro inhibitor of AACT/HMGS and might compete for the acetyl- CoA-binding site.

That in plants we might have an unusual system of HMG-CoA formation is indicated by the hitherto un- known cofactor requirement. The apparent coopera- tion between PQQ (as the most efficient quinone found so far) and Fen as enzyme activators leads us to postulate that there exists a flux of electrons from Fen to quinone, probably with semiquinone-radicals as in- termediates. Recently we have shown that the activa- tion by Fe”, measured in the absence of a quinone cofactor, was efficiently blocked by the radical scav- enger hydroxyurea 181, which gave us a first clue to assuming a radical mechanism as being involved in HMG-CoA formation. In vitro ESR measurements

showed that an as yet unidentified organic radical can be detected during the first 4 minutes of the enzymatic reaction. Neither in the absence of PQQ and of sub- strate in the test-system, nor in the absence of purified AACT/ HMGS was this radical detected (T. Weber, P. Such and T.J. Bach, unpublished observations). It is

94 T. Weber, T.J. Bach / Biochimica et Biophysics Acta 1211 (1994) 8.5-96

reasonable to assume that this radical plays an impor- tant role in the proton abstraction from the acetyl-CoA, which reacts as a nucleophil in the first partial reaction of HMG-CoA synthesis, thereby facilitating the other- wise energetically unfavorable Claisen condensation. In this mechanism, in a catalytic manner, the redox pair Fe”/quinone would act via redox-cycling. Although the involvement of PQQ or other amino acid-derived quinone cofactors in redox-cycling has been described (for review see Refs. 27-291, to our knowledge this would be the first example of the formation of a carbon-carbon bond supported by such a mechanism. Though the proton to be abstracted from acetyl-CoA during the first partial AACT reaction might already be sufficiently acidic through the neighbourhood of the thioester bonding or due to the microenvironment of enzyme protein at the active site even in the absence of a quinone radical (see Ref. 301, the radical might well shift the equilibrium to the formation of (enzyme- bound) acetoacetyl-CoA. For the carboxylation of glu- tamate by a vitamin Kl-dependent carboxylase within the reaction cascade of blood clotting, where it is necessary to abstract a proton, it was proposed that the reaction of vitamin Kl with oxygen leads to the forma- tion of a strong basic group responsible for proton abstraction [31]. But for the same reaction a radical mechanism has also been proposed [32]. Rather high concentrations of hydroxyurea, a radical scavenger, in- hibited AACT/HMGS in vitro [8], but similar concen- trations (lo-100 mM) had been used to disactivate Fen dependent ribonucleotide reductase from Scenedesmw oZ@us containing a tyrosin radical [19]. Moreover, addition of 10 PM Fen under aerobic conditions led there to a spontaneous reactivation of the enzyme [191.

Of thiolase from animal tissue or from yeast it is known that the equilibrium lies far on the side of thioclastic cleavage of acetoacetyl-CoA to 2 acetyl-CoA in the presence of HS-CoA (see Ref. 6 and literature cited therein), while for HMGS it lies far on the side of HMG-CoA formation, most likely driven by the ener- getically favorable hydrolysis of a thioester bond. Thus, in the overall reaction pathway, the presence of Fe”/ PQQ (or any similar quinone cofactor having a suitable redox potential, yet to be defined considering the pre- sent discussions as to PQQ as cofactor of eukaryotic quinoproteins [20,29;33]) could shift the equilibrium further to the side of HMG-CoA formation.

It is known that under aerobic conditions chelated Fe is subjected to a continuous redox cycling with consumption of reduced glutathion and ascorbic acid [17]. Fen reacts with oxygen to yield Fe’” and the superoxide radical 0,. At higher (physiological) pH values the spontaneous reaction to H,O, is suppressed and is catalyzed by superoxide dismutase 1171. The 0, radical can be trapped by quinones 1341 and the exis- tence of a radical (semi-quiuone) form of PQQ

(PQQH’) is known [28]. The formation of the superox- ide radical in the presence of Fe”/EDTA and oxygen could proceed directly at the enzyme-quinone complex: PQQ possesses several potential binding sites for metal cations [27,28]. The observation that under semi- anaerobic conditions, viz. in the presence of glucose and glucose oxidase in the assay mixture, the AAcT/ HMGS reaction is partially impaired (data not shown), further supports the hypothesis of peroxide radical formation to be involved in the reaction mechanism.

Kinetic analysis of AACT/HMGS activation by Fe”/PQQ indicates possibly the induction of some conformational change by the quinone cofactor, lead- ing to an apparent increase in affinity towards the substrate acetyl-CoA. This could have some function in vivo, where the availability of acetyl-CoA under a given physiological condition might become limiting to sup- ply the isoprenoid pathway. It is also possible that the release of product is facilitated in the presence- of Fe”/PQQ, due to some conformational effect, since removal of HMG-CoA by exogenously added- HMGR (activity measured as incorporation of [I- “C]acetyl- CoA into mevalonate) actually increases the reaction rate as compared to the control ([1-‘4C]acetyl-CoA incorporated into HMG-CoA) when the cofactors are absent, while this additional activation is much less prominent in the presence of Fe”/PQQ (Table 21,

The determination of Fe by extraction of plant tissue with I N HCl led to values of 10.5 to 33 gg Fen per g fresh weight [35]. If we take a water content of 90% in tissue of radish seedlings, a value of 10.5 pg would translate into a concentration of about 170 PM; more than three times higher than that needed to lead to a double in vitro activation, not considering-that within the cell, due to compartmentalization, much higher concentrations could occur. In plant tissue natu- ral chelators such as citrate [17] or ATP f36] are always sufficiently available.

Of course, although only one band was visible in silver-stained SDS gels, a contamination of less than l%, e.g., by ‘thiolase’ protein could not be detected in this way. However, in earlier experiments using par- tially purified radish AACT/HMGS, we added thio- lase partially purified from a yeast strain (kindly pro- vided by F. Karst, Poitiers, France) which overex- presses the AACT gene from Succharc~~& uuatiti (see Ref. 37). In no case we could see any difference to measurements done in the absence of additional thio- lase protein (unpublished observations). Thus, it seems very unlikely that we only deal with a contamination not detectable otherwise. Furthermore, the stimulat.ion by Fen/PQQ becomes significant only with largeiy purified enzyme and is hardly visible in crude extracts (S. Zeiler and T.J. Bach, unpublished observations). in our initial attempts of distinguishing between AACT and HMGS, using crude solubilized enzyme prepara-

T. Weber, TX Bach /Biochimica et Biophytia Acta 1211 (1994) 85-96 95

tions from radish membranes, we applied the tech- niques as described by Stewart and Rudney 1381 char- acterizing yeast AACT. However, neither by trypsin digestion nor by sodium borohydride treatment it was possible to exclusively disactivate AACT and thus to clearly dissect AACT from HMGS activity (Weber and Bach, ~pub~hed observations). Unfo~ately, due to substrate inhibition at concentrations higher than 50 PM, as yet it was not possible to monitor by NMR techniques the in vitro conversion of substrate to HMG-CoA using ‘H- and/or 13C-labeled acetyl-CoA. Minimum concentrations of acetyl-CoA for obtaining clear signals exceeded several times this limit. Further- more, the quantities of purified enzyme needed are difficult to prepare. However, such an approach would be feasible after isolation of a corresponding gene and heterologous overexpression as has been shown possi- ble with radish HMGR [ll].

The localization of AACT and HMGS activity on only one protein allows for a directed flux of carbon units into the isoprenoid pathway, not excluding that the sequel enzyme HMGR, in vivo, forms a close complex with AACT/ HMGS. In view of the numerous enzymes ~rnpe~g for the central substrate acetyl-CoA in plants [6f, such a substrate channeling would make functional sense. This could be one of the reasons for this different mechanism of HMG-CoA synthesis in Raphunzu satiuus and probably in other plants as well, in comparison to the well-known situation in yeast and meals. Qn the other hand, given the fact that the membrane fraction used to isolate AACT/ HMGS from radish is also a major source for HMG-CoA lyase &IMGL, see Ref. 39) catalyzing the cleavage of HMG- CoA to acetoacetate plus acetyl-CoA in a retro-Claisen ~ndensation reaction [40,41], although less active than ~~/~GS when measured in vitro, its presence could indicate that in plants AACI’/HMGS plays a role in a ketogenic cycle. However, to our knowledge there does not exist clear information as to the metabolic fate in plants of acetoacetate generated by the HMGL reaction [6]. There also exists evidence of the occurrence of a mevalonate shunt pathway in higher plants [42] with HMG-CoA as an intermediate, as it is most likely true for the degradation of odd-numbered amino acids. At present the intracellular compartmen- tation of those intervening and/or scavenging path- ways is not yet known, nor is their regulation. Prelimi- nary results of immunocytochemical studies with roots of dark-grown, mevinoliu-treated radish seedlings using antibodies raised against AACT/HMGS provide fur- ther evidence of a membrane-associated localization of this enxyme [43].

It remains the problem of whether PQQ does really occur in plants and whether it represents the “natural” cofactor of the AACT/HMGS reaction, A recent pa- per [441 reports the presence of PQQ in pistils and

pollen grains of hiier plauts, which suggests that its presence might not be limited to mi~roorgani~s. But we would not exclude that another plant-specific quinone could play a similar role. Even in view of the nearly unlimited number of quinone derivatives which occur in plants we have now started to test a great variety of natural or synthetic ~mpo~ds in order to more closely define the structural requirements of en- zyme activation.

Our current attempts are directed towards cloning the gene&> encoding radish AACT/HMGS by various strategies such as screening of cDNA expression li- braries with stifles or by implementation analysis of suitable yeast mutants.

5. Acknowledgements

We wish to thank Mrs. Sabine Zeiler for expert technical assistance and Professor H.K. Lichtenthaler for a generous gift of halogenated naphthoquinone derivatives and for his steady interest in the progress of thii work. We are grateful to Professor Robert H. Abeles, Brandeis University and to Professor Janos Retey, Karlsruhe, for helpful discussions and to Mr. Philip Jackson for reading the English manuscript. This work was supported by the Deutsche Forschungsge meinschaft (Grant No. Ba 871/2-3, to T.J.B.). Essen- tial results of this study have been presented at the following international meetings: (a) ACS/ FASEB Symposium, Biosynthesis and Utilization of Iso- prenoids (Reductase V), April 18-20, 1991, Atlanta, Georgia; (b) lOti International Symposium on the Metabolism, Structure and Utilization of Plant Lipids, April 27-May 2, 1992, Jerba, Tunisia; (c) Gordon Conference on Quinone and Redoxactive Amino Acid Cofactors, July 5-10, 1992, Andover, New Hampshire.

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