[13] Acetyl-CoA Synthetases I and II from Pyrococcus furiosus

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[13]

2 units/mg, respectively. The specific activities of IOR and K G O R are about 10-fold lower than that of VOR. These data can be used to give a reasonable estimate of the relative amounts of these proteins within the cells, as the specific activities of the purified enzymes are comparable (20 and 50 U/mg). The four enzymes vary in their affinities for CoASH (K,, of 17-110/~M) and for ferredoxin, the physiological electron carrier (Km of 8-94/xM, see Table III). The physiological significance of these differences, if any, is not known. None of the enzymes are able to couple 2-keto acid oxidation to the reduction of NAD or NADP, which is consistent with ferredoxin being the physiological electron acceptor. In addition to the oxidative decarboxylation of pyruvate to produce acetyl-CoA, P O R from P. furiosus has been shown to catalyze the decarboxylation of pyruvate to generate acetaldehyde in a CoA-dependent reaction. 34 The apparent Km values for CoASH (0.11 mM) and pyruvate (1.1 mM) in the nonoxidative decarboxylation reaction are very similar to those determined previously for pyruvate oxidation. The other three KORs probably also catalyze this reaction, and it has been proposed that this is of some significance because these enzymes would generate various aldehydes from the transaminated forms of amino acids. 34 However, this has yet to be confirmed by in vivo analyses. Acknowledgment This research was supported by grants from the Department of Energy. 34K. Ma, A. Hutchins, S. S. Sung, and M. W. W. Adams, Proc. Natl. Acad. Sci. U.S.A. 94, 9608 (1997).

[13] A c e t y l - C o A S y n t h e t a s e s

from

I a n d II

Pyrococcusfuriosus

B y ANDREA M. HUTCHINS, XUHONG MAI, a n d MICHAEL W. W. ADAMS

Introduction Acetate and acetyl-CoA are important intermediates in microbial metabolism. Fatty acids, polysaccharides, and proteins are all broken down into acetyl-CoA units, a requisite step prior to energy generation. 1 Acetyl1R. K. Thauer, K. Jungerrnann, and K. Decker, Bacteriol. Rev. 41, 100 (1977).

METHODS IN ENZYMOLOGY, VOL. 331

Copyright © 200i by Academic Press All rights of reproduction in any form reserved. 0076-6879/00 $35.00

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CoA is used as a building block in the synthesis of various cell components or it is transformed to acetate as an end product of certain fermentative pathways. The conversion of acetyl-CoA to acetate is, therefore, a key step in general metabolism. In most bacteria, this transformation is catalyzed by two enzymes: phosphoacetyltransferase and acetate kinase. These catalyze the reactions shown in Eqs. (1) and (2), respectively. Acetyl-CoA + Pi--~ acetyl phosphate + CoA Acetyl phosphate + ADP ~ acetate + ATP

(1) (2)

In hyperthermophilic archaea, such as Pyrococcusfuriosus, however, acetylCoA is converted to acetate in a single step, which is carried out by an enzyme known as ADP-dependent acetyl-CoA synthetase [EC 6.2.1.13; acetate-CoA ligase (ADP-forming)] [Eq. (3)]. 2-4 Acetyl-CoA + ADP + Pi --, acetate + CoA + ATP

(3)

Acetyl-CoA synthetase (ADP-dependent) has so far been found only in certain archaea, including hyperthermophiles and halophiles, and in the eukaryotic protists Entamoeba histolytica and Giardia lamblia. 5,6 An analogous enzyme is present in some bacteria] Termed acetyl-CoA synthetase (AMP-forming, EC 6.2.1.1; acetate-CoA ligase), it couples the conversion of acetate to acetyl-CoA with the generation of AMP and pyrophosphate from ATP [Eq. (4)]. Acetate + CoA + ATP--~ acetyl-CoA + AMP + PP~

(4)

In contrast to the archaeal ADP-dependent enzyme, the bacterial acetylCoA synthetase has a kinetic preference for catalyzing the activation of acetate to acetyl-CoA, rather than the production of acetate. Formation of the high-energy thioester bond of acetyl-CoA requires the hydrolysis of pyrophosphate as a driving force, so AMP, rather than ADP, is the product of the reaction [Eq. (4)]. Two distinct enzymes with ADP-dependent acetyl-CoA synthetase activity have been purified from cell-free extracts of P. furiosus, 3 an organism that grows by fermenting both sugars and peptides. 8 These enzymes convert the acetyl-CoA produced by the fermentative pathways into acetate with the concomitant production of ATP. Acetyl-CoA is produced by the oxidative 2 T. Schafer and P. Sch6nheit, Arch. Microbiol. 155, 366 (1991). 3 X. Mai and M. W. W. Adams, J. Bacteriol. 178, 5897 (1996). 4 j. Glasemacher, A.-K. Bock, R. Schmidt, and P. Sch~nheit, Eur. J. Biochem. 244, 561 (1997). s T. Schiller and P. Schrnheit, Arch. Microbiol. 159, 72 (1993). ~'L. B. Sanchez and M. MUller, FEBS Lett. 378, 240 (1996). v G. G. Preston, J. D. Wall, and D. W. Emerich, Biochern. J. 267, 179 (1990). G. Fiala and K. O. Stetter, Arch. Microbiol. 145, 56 (1986).

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decarboxylation of pyruvate by pyruvate ferredoxin oxidoreductase (POR). 9'1° However, the two enzymes, which are termed acetyl-CoA synthetase (ACS) I and II, differ in their substrate specificity? ACS I uses isobutyryl-CoA as a substrate as well as acetyl-CoA, but it will not utilize phenylacetyl-CoA or indoleacetyl-CoA. ACS II, however, utilizes all four of these substrates, but neither enzyme uses succinyl-CoA as a substrate. This wide substrate specificity suggests that these enzymes have functions in addition to the production of acetate from acetyl-CoA. The other CoA derivatives are thought to be derived from the fermentation of amino acids. 11 Specifically, peptide-derived amino acids are transaminated to the corresponding 2-keto acids, each of which is converted to the CoA derivative by three additional 2-keto acid oxidoreductases found in P. furiosus, termed IOR, 12 VOR, 13 and KGOR. 14 IOR is specific for the transaminated products of aromatic amino acids, whereas V O R utilizes 2-keto acids derived from the amino acids valine, isoleucine, leucine, and methionine, and 2-ketoglutarate is the only known substrate of KGOR. Hence, although neither ACS I or ACS II converts succinyl-CoA, the product of the K G O R reaction, both enzymes are able to utilize the products of the P O R and V O R reactions as substrates, and only ACS II can use the aromatic derivatives produced by IOR. 11 This article describes the methods used to assay and purify ACS I and ACS II from P. furiosus, along with some of their molecular and catalytic properties.

Assay Methods for ACS I and ACS II The two ACS isoenzymes can be assayed in either direction [Eq. (3)] by the production of the acid or of the CoA derivative. Production of the CoA derivative [reverse reaction in Eq. (3)] is followed by measuring the amount of phosphate formed. This assay is suitable for kinetic analyses. Two assays are used to measure acid production [forward reaction in Eq. (3)]. The routine assay used in our laboratory employs a coupled system involving a thermostable 2-keto acid oxidoreductase, but this assay cannot be used for some kinetic analyses as the CoA derivative is regenerated. 9 G. J. Schut, A. L. Menon, and M. W. W. Adams, Methods Enzymol. 331 [12] (2001) (this volume). a0 j. M. Blarney and M. W. W. Adams, Biochim. Biophys. Acta 1161, 19 (1993). 11 M. W. W. A d a m s and A. Kletzin, Adv. Prot. Chem. 48, 101 (1996). 12 X. Mai and M. W. W. Adams, J. Biol. Chem. 269, 16726 (1994). 13 j. Heider, X. Mai, and M. W. W. Adams, J. Bacteriol. 178, 780 (1996). 14 X. Mai and M. W. W. Adams, J. Bacteriol. 178, 5890 (1996).

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The second assay involves the direct measurement of CoA production from the CoA derivative.

Coupled Assay for Acid Production from CoA Derivative In this assay the CoA derivative that serves as the substrate for ACS is generated as a product of reaction catalyzed by the corresponding 2keto acid oxidoreductase. These enzymes catalyze the general reaction shown in Eq. (5), where Mgred(ox) is the oxidized (reduced) form of the artificial electron carrier methyl viologen. For the ACS assay, the oxidoreductase reaction is carried out with limiting amounts of CoA. The continuous reduction of MV is only possible if CoA is regenerated by ACS as it converts the CoA derivative to the acid [Eq. (6)]. Hence, ACS activity can be measured by following the reduction of MV, which is performed R C O C O O H + 2MVox + CoA ~ R C O C o A + CO2 + 2MVred R C O C o A + A D P + Pi ~ R C O O H + CoA + ATP

(5) (6)

spectrophotometrically. Note that reduced MV autoxidizes rapidly in air and so the assay must be carried out under anaerobic conditions. 9 This assay is a simple and quick method for detecting ACS activity, although it requires the appropriate thermostable oxidoreductase. P O R is used in the routine assay to generate acetyl-CoA for ACS I, whereas IOR generates indoleacetyl-CoA in the assay of ACS II. Assays are carried out in serum-stoppered cuvettes under an argon atmosphere where all reagents are degassed and flushed with argon prior to u s e Y 5 For ACS I, the 2-ml reaction mixture contains 10 mM pyruvate, 5 mM MgCI2, 0.4 mM thiamin pyrophosphate (TPP), 5 mM MV, 10 mM KzHPO4, and 50 m M N-(2-hydroxyethyl)piperazine-N'-3-propanesulfonic acid (EPPS) buffer, pH 8.4. The cuvette is placed in the thermostated holder of a Spectronic 501 spectrophotometer (Fisher Scientific, Atlanta, GA) and heated to 80 ° using a circulating water bath. After the addition of 0.025 mM CoA, 40 I~g P. furiosus POR and a sample containing ACS I, 1 m M A D P is added to start the reaction. The reduction of colorless oxidized MV to the blue-colored reduced form is measured at 600 rim. A molar absorbance for reduced MV of 12,000 M -1 cm -1 is used to calculate ACS I activity, where 1 unit equals the reduction of 2/zmol of MV/min. This is equivalent to 1/~mol of the corresponding acid produced/rain. ACS II activity is measured in the same manner as ACS I except that 10 mM indolepyruvate and 40/~g I O R replace pyruvate and POR, respectively. 1.~M. F. J. M. Verhagen.A. L. Menon, G, J. Schut, and M. W. W. Adams, Methods Enzymol. 33o [31 (2001).

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Coupled Assay for Acid Production from CoA Derivative This assay depends on measuring the CoA produced from the CoA derivative [Eq. (6)]. The reaction mixture (2.0 ml) contains the CoA derivative (acetyl-CoA or isobutyryl-CoA for ACS I, and acetyl-CoA, isobutyrylCoA, or phenylacetyl CoA for ACS II) (0.1 mM), ADP (2 mM), KzHPO4 (10 mM), MgCI2 (2 mM), and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB; 0.1 mM) in 50 mM EPPS buffer, pH 8.4. The activity is measured spectrophotometrically at 80° under anaerobic conditions as described earlier except that absorbance changes are monitored at 412 nm. An extinction coefficient of 13,600 M -1 cm i was used for the DTNB derivative. Activities are calculated as micromoles of CoA produced per minute.

Formation of CoA Derivative from Acid A discontinuous assay system is used to measure the production of the CoA derivative [reverse of Eq. (6)]. The CoA derivative, phosphate, and ADP are formed in the first step at 80°. The second step involves measuring the amount of phosphate produced, which is carried out at either 37° or 45 °. The phosphate detection method used is modified from that reported previously. 16 This is based on the reaction between molybdate and phosphate under acidic and reducing conditions to form a blue polymeric complex [PMo12040]. For ACS I, the 0.5-ml assay mixture contains 10 mM acetate, 10 mM MgCI2, and 50 mM EPPS buffer, pH 8.4. In the assay of ACS II, 10 mM indoleacetate is used instead of acetate. The mixture is incubated at 80° and the enzyme sample and 2 mM ATP are added to start the reaction. The reaction is stopped after 3 rain by adding 0.1 ml of 6.0 M HzSO 4 and the mixture is placed at 23 °. Note that approximately 10% of the ATP is hydrolyzed abiotically in this reaction so appropriate control assays without enzyme must be carried out. To determine the amount of phosphate produced, remove 0.15 ml of the assay mixture and add it to 0.15 ml H20. To this add 0.7 ml of an ascorbate/molybdate solution freshly prepared by mixing stock solutions of 0.42% (w/v) ammonium molybdate in 1.0 M H2804 and 10% (w/v) ascorbic acid in a 1 : 6 ratio. The 1-ml mixture is incubated at either 45 ° for 25 min or 37° for 60 min and the absorbance is measured at 820 nm. The molar absorption coefficient for heteropoly blue is 26,000 M -1 cm -1.

16H. Hasegawa, M. Parniak, and S. Kaufman, AnaL Biochem. 120, 360 (1982).

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Purification of ACS I a n d ACS II

Pyrococcus furiosus (DSM 3638) is obtained from the Deutsche Sammlung yon Mikroorganismen, Germany. It is grown routinely at 90 ° in a 600-liter fermentor with maltose as the carbon source as described previously) 5'17 ACS I and ACS II are purified from the same cell-free extract of P. furiosus. 3 In fact, the same extract can be used to purify several other enzymes and proteins from this organism. 15 Many of these proteins are oxygen sensitive and strictly anaerobic and reducing conditions are required to minimize loss of activity during purification. Whereas ACS l and ACS II are not oxygen sensitive, and could be purified aerobically, they are also routinely purified anaerobically. All of the purification steps are carried out at 23 °, and the buffers used throughout are repeatedly degassed and flushed with argon, contain 2 mM sodium dithionite (DT) and 2 mM dithiothreitol (DTT), and are kept under a positive pressure of argon. ACS I and ACS II are purified routinely from 500 g (wet weight) of ceils at 23 ° under strict anaerobic conditions. The procedures to prepare the cell-free extract and to carry out the first chromatography step are described elsewhere in this volume. 15 In brief, cells are thawed in (1 g per 3 ml) 50 mM Tris-HC1, pH 8.0, containing 2 mM DT, 2 mM DTT, and 0.5 /~g/ml DNase I. After incubation at 37 ° for 1 hr and centrifugation at 30,000g for 1 hr at 4 °, the cell-free extract is loaded onto a column (10 × 20 cm) of DEAE-Sepharose FF (Pharmacia Biotech) equilibrated with 50 mM Tris-HC1 containing 2 mM DT and 2 mM DTT. The extract is diluted threefold with the buffer as it is loaded. The bound proteins are eluted with a linear gradient (15 liters) from 0 to 0.5 M NaC1 in the equilibration buffer and 125-ml fractions are collected. ACS II activity is measured routinely by the IOR-coupled assay system, which elutes as 70 to 140 mM NaC1 is applied to the column. The activity of ACS I is determined routinely by the POR-linked assay and elutes as 160 to 200 mM NaCI is applied. The two enzymes are, therefore, well separated by this chromatography step and are further purified independently. ACS I

Hydroxyapatite Chromatography. Fractions with ACS I activity from the DEAE-Sepharose column are directly loaded onto a column (5 × 10 cm) of hydroxyapatite (Bio-Rad, Hercules, CA) equilibrated with 50 m M 17F. O. Bryant and M. W. W. Adams, J. Biol. Chem, 264, 5070 (1989).

164

[13]

ENZYMES OF CENTRAL METABOLISM

Tris-HC1, pH 8.0, containing 2 mM DT and 2 mM DTT (hereafter referred to as buffer A). The protein is eluted at 3 ml/min with a 1.2-liter linear gradient from 0 to 200 mM potassium phosphate in buffer A. ACS I elutes as 70 mM phosphate is applied to the column and is collected in 40-ml fractions. Phenyl-Sepharose Hydrophobic Interaction Chromatography. ACS Icontaining fractions are pooled and diluted with an equal volume of 2.0 M (NH4)2SO4in buffer A. This is applied directly to a column (3.5 x 10 cm) of phenyl-Sepharose (Pharmacia Biotech, Piscataway, N J) equilibrated with 1.0 M (NH4)2SO4in buffer A. A 600-ml decreasing linear gradient from 1.0 to 0 M (NH4)2804is applied to the column at a flow rate of 6 ml/min to elute the protein. Fractions of 45 ml are collected. ACS I begins to elute as 520 mM (NH4)2504is applied. Superdex 200 Gel Filtration Chromatography. Fractions containing ACS I activity are concentrated to approximately 12 ml by ultrafiltration using a PM30 membrane (Amicon, Bedford, MA) and applied to a column (6 x 60 cm) of Superdex 200 (Pharmacia Biotech) equilibrated with buffer A containing 200 mM NaCI. The flow rate is 3 ml/min and 10-ml fractions are collected. Those containing ACS I activity above 62 units/mg are analyzed separately by SDS-PAGE. Those judged pure are combined, concentrated by ultrafiltration, and stored at - 8 0 °. Table I shows the results from a typical purification of ACS I. Approximately 140 mg of pure protein is obtained with a specific activity of 65 units/mg in the POR-linked coupled assay. The overall recovery of activity from the cell-free extract is only about 12% but note that only about 35% of the activity is recovered after the first chromatography step. This is probably due to an overestimation of ACS I activity in the cell-free extract. Several factors present could interfere with the coupled assay system, including many oxidoreductase-type enzymes that reduce MV. 15 Such a conclusion is supported by a similar loss in activity of ACS II after the first TABLE I PURIFICATION OF Pyrococcusfuriosus ACETYL-CoA SYNTHETASE I

Step

Activity (units)

Protein (rag)

Specific activity ~ (units/mg)

Recovery (%)

Purification (-fold)

Extract DEAE-Sepharose HAP Phenyl-Sepharose Superdex 200

74,800 26,100 21,800 16,000 9,300

27,000 3,900 2,570 360 144

2.8 6.6 8.0 45.0 65.0

100 35 29 21 12

1 2 3 16 23

a ACS I activity was determined using the POR-linked assay.

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chromatography step (see later) and by the fact that neither enzyme showed significant activity losses in subsequent chromatography steps (Tables I and II).

ACS H Hydroxyapatite Chromatography. Fractions from the initial D E A E Sepharose FF column containing ACS II activity are applied directly to a column (5 x 10 cm) of hydroxyapatite (Boehring Diagnostics) equilibrated with buffer A. The protein is eluted at 3 ml/min with a 1.2-liter linear gradient from 0 to 200 mM potassium phosphate in buffer A. ACS II elutes as 65 mM phosphate is applied to the column and is collected in 40ml fractions. Phenyl-Sepharose Hydrophobic Interaction Chromatography. Fractions containing ACS II activity are pooled and diluted with an equal volume of 2.0 M (NH4)2SO4 in buffer A. This is applied directly to a column (3.5 x 10 cm) of phenyl-Sepharose (Pharmacia Biotech) equilibrated with 1.0 M (NH4)2SO4 in buffer A. A 600-ml decreasing linear gradient from 1.0 to 0 M (NH4)2SO4 is applied to the column at a flow rate of 6 ml/min to elute the protein. Fractions of 20 ml are collected. ACS II begins to elute as 470 mM (NH4)2SO4 is applied. Superdex 200 Gel-Filtration Chromatography. Those fractions containing ACS II activity are concentrated to approximately 5 ml by ultrafiltration (PM30 membrane, Amicon) and applied to a column (6 × 60 cm) of Superdex 200 (Pharmacia Biotech) equilibrated with buffer A containing 200 m M NaCl at 3 ml/min. Fractions of 10 ml are collected. Those with ACS II activity above 30 units/mg are separately analyzed by S D S - P A G E and those judged pure are combined, concentrated by ultrafiltration, and stored at - 8 0 °. TABLE II PURIFICATIONOFPyrococcusfuriosus ACETYL-CoASYNTHETASE1I

Step

Activity (units)

Extract DEAE-Sepharose HAP Phenyl-Sepharose Superdex 200

3430 1800 1160 908 600

Protein (mg) 27,000 838 372 32 20

Specific activity" (units/mg) 0.1 2.2 3.1 28.4 30.0

"ACS II activitywas determined using the IOR-linked assay.

R e c o v e r y Purification (%) (-fold) 100 54 34 26 17

1 17 24 218 23l

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The results of a typical purification procedure are shown in Table II. Approximately 20 mg of pure protein is obtained with a specific activity in the IOR-linked assay of 30 units/mg. Properties of ACS I and ACS II ACS I and ACS II have very similar molecular properties. 8 Their molecular masses are about 140 kDa as judged by gel filtration and both appear to be heterotetramers (a~fla) of two different subunits with masses of 45 and 23 kDa as judged by SDS-PAGE. They do not seem to contain any cofactor as neither protein exhibited absorption in the visible region of the spectrum and neither contained iron or other metals, such as copper, zinc, or magnesium. That ACS I and ACS II are distinct enzymes was shown by the amino-terminal sequences for their two subunits, which were similar but not identical. These were used to identify the genes encoding ACS I and ACS II in the genome database. The two genes (acdAI and acdBI) encoding the two subunits (a and r , respectively) of ACS I correspond to proteins with molecular weights of 49,964 (a) and 25,878 (fl),18 whereas those for ACS II (acdAH and acdBll) correspond to proteins with molecular weights of 49,259 (a) and 26,442 (fl). The two a and the two fl subunits show 49 and 56% amino acid sequence identity, respectively. Genes encoding the four subunits are separated from each other on the genome, and those encoding subunits Ia and Ifl appear to be transcribed individually. In contrast, the gene encoding subunit IIo~ seems to be part of an operon that also contains the genes encoding the two subunits of IOR, whereas the gene encoding subunit lift appears to be cotranscribed with a putative gene, the product of which would have a sequence similarity (47% identical) to that of subunit lot. It is interesting to note that the genome has two other putative genes that would encode proteins with high sequence similarity (-40% identity) to the Io~ subunit. The function of these three ACS-like genes is not known at present. The genes encoding ACS I have been expressed individually in Escherichia coli, and the properties of the reconstituted holoenzyme are virtually indistinguishable from those of the native enzyme purified from P. furiosus. 18 Neither ACS I nor ACS II shows any loss of activity after exposure to 02 (air) for 24 hr, and both enzymes could presumably be purified aerobically without deleterious effects.3 Both enzymes are also very thermostable. The times required for a 50% loss of activity on incubating ACS I (0.4 mg/ml) and ACS II (0.5 mg/ml) at 80° in EPPS buffer (pH 8.0) are 18 and 8 hr, respectively. Using the POR-linked assay, ACS I is virtually inactive at a8M. Musfeldt,M. Selig, and P. Sch0nheit,J. Bacteriol. 181,5885 (1999).

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ambient temperature and shows a dramatic increase in activity above 70 ° with an optimum above 90° (at pH 8.0). Virtually identical results are obtained with ACS II using the IOR-linked assay. With both ACS I and ACS II in the acid formation reaction [forward reaction of Eq. (6)], ADP and phosphate can be replaced by GDP and phosphate, but not by CDP and phosphate or AMP and pyrophosphate. -~ The apparent Km values for ADP, GDP, and phosphate are approximately 150, 132, and 396 IxM, respectively, for ACS I (using acetyl-CoA) and 61, 236, and 580/xM, respectively, for ACS II (using indoleacetyl-CoA). With ADP and phosphate as substrates, the apparent Km values for acetyl-CoA and isobutyryl-CoA are 25 and 29/xM, respectively, for ACS I and 26 and 12/xM, respectively, for ACS II. With ACS II, the apparent Km value for phenylacetyl-CoA is 4/zM. 3 Note that ACS I does not use this CoA derivative as a substrate, and neither enzyme utilizes succinyl-CoA. Both enzymes also catalyze the reverse reaction shown in Eq. (6), the ATP-dependent formation of the CoA derivatives. Their specific activities are similar to those measured in acid formation 3 as are their substrate specificities. Thus, both enzymes utilize acetate and isobutyrate, but only ACS II utilizes phenylacetate and indoleacetate. However, from the apparent K,, values, the affinities of both enzymes for the acids are much lower (by at least an order of magnitude) than they are for the CoA derivatives. For example, for ACS I (and for ACS II), the values for acetate and isobutyrate are 1.1 (10.7) and 0.46 (5.8) mM, respectively, and ACS II exhibitis Km values for phenylacetate and indoleacetate of 0.77 and 2.0 mM, respectively. These data are consistent with the proposal that both enzymes function in acid production as part of the fermentation pathways of P. furiosus. 3"11It is also possible that, under conditions of nutrient limitation, ACS I and ACS II catalyze the reverse reaction and provide carbon skeletons for amino acid biosynthesis, 13 although at present there is no evidence to support this. Acknowledgment This research was supported by grants from the Department of Energy and the National Science Foundation.