ARCHIVES

OF BIOCHEMISTRY

Vol. 299, No. 1, November

AND

BIOPHYSICS

15, pp. 77-82, 1992

Evidence for an Essential Histidine Residue in 4SLimonene Synthase and Other Terpene Cyclases’ Jean I. M. Rajaonarivony,’

Jonathan

Gershenzon, John Miyazaki,3 and Rodney Croteau4

Institute of Biological Chemistry, and Graduate Program in Plant Physiology, Washington State University, Pullman, Washington 99164-6340

Received May 7, 1992, and in revised form July 9, 1992

(4S)-Limonene synthase, isolated from glandular trichome secretory cell preparations of Mentha X piperita (peppermint) leaves, catalyzes the metal ion-dependent cyclization of geranyl pyrophosphate, via 3S-linalyl pyrophosphate, to (-)-(4S)-limonene as the principal product. Treatment of this terpene cyclase with the histidinedirected reagent diethyl pyrocarbonate at a concentration of 0.25 mM resulted in 50% loss of enzyme activity, and this activity could be completely restored by treatment of the preparation with 5 mM hydroxylamine. Inhibition with diethyl pyrocarbonate was distinguished from inhibition with thiol-directed reagents by protection studies with histidine and cysteine carried out at varying pH. Inactivation of the cyclase by dye-sensitized photooxidation in the presence of rose bengal gave further indication of the presence of a readily modified histidine residue. Protection of the enzyme against inhibition with diethyl pyrocarbonate was afforded by the substrate geranyl pyrophosphate in the presence of Mn’+, and by the sulfonium ion analog of the linalyl carbocation intermediate of the reaction in the presence of inorganic pyrophosphate plus Mn’+, suggesting that an essential histidine residue is located at or near the active site. Similar studies on the inhibition of other monoterpene and sesquiterpene cyclases with diethyl pyrocarbonate suggest that a histidine residue (or residues) may play an important role in catalysis by this class of enzymes. ‘~1 1992

Academic

Press.

Inc.

i This investigation was supported in part by grants from the National Institutes of Health (GM-31354) and the Department of Energy (DEFG0688ER13869) and by Project 0268 from the Washington State University Agricultural Research Center, Pullman, WA 99164. ’ AFGRAD Fellow of the African-American Institute. Present address: Department of Botany, University of British Columbia, Vancouver, BC Canada. ’ Present address: Abbott Laboratories, Abbott Park, IL 60064. * To whom correspondence should be addressed. Fax: (509) 335-7643. 0003.9861/92

$5.00 1992 by Academic Press, of reproduction in any form

Copyright Q All

rights

Monoterpene synthases (cyclases) catalyze the divalent metal ion-dependent conversion of geranyl pyrophosphate to a variety of monocyclic and bicyclic carbon skeletons (1). Because these enzymes catalyze the committed step of monoterpene biosynthesis and function at the C,,branch point of the isoprenoid pathway, they are presumed to have an important role in the regulation of monoterpene formation (2). Detailed study of the cyclizations of geranyl pyrophosphate (3), and of related reactions in the sesquiterpene (4) and diterpene series (5), has supported a common electrophilic reaction mechanism for the construction of these terpenoid natural products. In many ways, this reaction mechanism resembles that involved in the prenyltransferase reaction (6) by which the corresponding acyclic Clo, C&, and CZOprecursors of the terpenoids are constructed (7). In the monoterpene cyclization reaction, the divalent metal ion is thought to assist in the initial ionization of the geranyl substrate (8) (Scheme I, 1) that is followed by isomerization to linalyl pyrophosphate, an enzymebound intermediate capable of cyclization (3). All monoterpene cyclases investigated to date are capable of catalyzing this required isomerization step, as well as the ensuing cyclization sequence, without the formation of free intermediates (3). Following isomerization, ionization of the tertiary allylic linalyl pyrophosphate intermediate (to 2) precedes cyclization to the cu-terpinyl cation (3) from which all other monoterpenes are formed by subsequent internal additions, hydride shifts, and rearrangements; in the formation of limonene only a simple terminating deprotonation is required (Scheme I). Extensive investigations of many such cyclizations have lead to the development of a general mechanistic model for this reaction type (3), and have defined the stereoelectronic requirements for substrate binding and ionization (9, 10). A number of monoterpene cyclases have been isolated and characterized but, beyond the demonstration of similar reaction parameters (pH optima, kinetic constants) and common properties (PI, hydrophobicity, molecular 77

Inc. reserved.

78

RAJAONARIVONY

ET AL.

OPP-M \

r * I

Geranyl pyrophosphate

0

(+I-3S-Linalyl pyrophosphate

0 I

OPP-M

OPP-M

.. d!

I 4

* (-)-4S-Limonene

2 SCHEME I. Pathway for the cyclization of geranyl pyrophosphate The sulfonium ion analog (4) of the linalyl cation (2) is illustrated

to (-)-4Slimonene. to the right.

weight), very little is known about the fine structure of these proteins, including active site organization and function (1, 3). An essential cysteine residue appears to be a universal feature of terpenoid cyclases, including limonene synthase (11); however, reports suggesting the possible involvement of other active site residues (methionine, arginine, serine, histidine) are scattered and not well-documented by substrate protection studies (3). In this paper, we describe the influence of diethyl pyrocarbonate and dye-sensitized photooxidation (with rose bengal) on the activity of limonene synthase from Mentha X piper&a (peppermint), and report similar studies with monoterpene cyclases of other herbaceous species. Based on the well-known specificity of these reagents (12-18), protection studies with substrates and analogs, and the uniformity of the effects observed, it is suggested that an active site histidine is a common feature of this enzyme class and plays an important role in cyclase catalysis. EXPERIMENTAL

PROCEDURES

Plants, substrates, and reagents. Mentha X piperita L. cv. Black Mitcham (peppermint) plants were grown from stolons under controlled conditions described previously (19). Immature, rapidly expanding leaves (0.5-1.0 cm in length) from the shoot apex of 3- to &week-old plants were used as the enzyme source. The preparation of [l-3H]geranyl pyrophosphate (5.55 GBq/mmol) (20, 21) and of the sulfonium ion analog of the linalyl cation ((R,S)methyl-[4-methylpent-3-en-1-yllvinyl-sulfonium perchlorate) (22) have been described. Unless otherwise specified, all other materials, reagents, and biochemicals were obtained from Bio-Rad Laboratories, Research Organics, Sigma Chemical Co., or Aldrich Chemical Co. Enzyme preparation and assay. The isolation of (-)-(4S)-limonene cyclase from sonicated peppermint glandular trichome secretory cell

1

4

OPP-M denotes the pyrophosphate-metal

ion complex.

clusters and the partial purification of this enzyme (to -40% purity) by anion-exchange chromatography on DEAE-cellulose (Whatman DE52) have been described (11, 19). The assay has also been described in detail elsewhere (1, 11, 19) and involves the metal ion-dependent cyclization of [1-‘Hjgeranyl pyrophosphate to the olefin limonene, followed by solvent extraction, purification by column chromatography, and aliquot counting. Product identity was routinely verified by radio-gasliquid chromatography (19, 23), and protein concentration was determined by the method of Bradford (24) using the Bio-Rad kit. Carbethoxylation and reversal experiments. Aliquots of the partially purified enzyme solution (generally 1 ml with -4 pg protein) in 15 mM potassium phosphate (pH 5.5-6.5), 15 mM Mopso (pH 6.5-7.0), or 15 mM Hepes (pH 7.0-8.0) buffer were preincubated for up to 45 min at 3O“C with 0.1 to 0.5 mM diethyl pyrocarbonate (ethoxyformic anhydride). To readjust the buffer to assay conditions (Mopso, pH 7.0, with 0.5 mM dithiothreitol and 10% (v/v) glycerol), the preincubated mixture was desalted into assay buffer by gel filtration (Econo-Pat 10 DG, Bio-Rad). The enzymatic reaction was then initiated by addition of 7 pM [l3H]geranyl pyrophosphate and 1 mM MnCl,. Diethyl pyrocarbonate was freshly diluted into cold ethanol for each experiment (12, 25), and the final concentration of ethanol in the enzyme reaction mixture never exceeded 5%. Control reactions with ethanol alone were included in all experiments, and the ethanol was shown to have negligible influence on cyclase activity. For reversal experiments (decarbethoxylation), the protein samples previously treated with diethyl pyrocarbonate (in Mopso, pH 7.0) were incubated with various concentrations of hydroxylamine (prepared as an aqueous stock solution of the hydrochloride adjusted to pH 7.0 with KOH) for 18 h at 4’C (12, 25). Excess hydroxylamine, residual diethyl pyrocarbonate, and other low molecular weight reactants were then removed, and the mixture was adjusted to assay conditions by gel filtration, as before, with initiation of the reaction by addition of substrate and metal ion. Control samples were similarly treated with hydroxylamine but contained no diethyl pyrocarbonate (only ethanol).

’ Abbreviations used: Mopso, 3-(N-morpholino))Z-hydroxypropanesulfonic acid, Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonicacid.

HISTIDINE

RESIDUES

Photooridattin. Photooxidation was carried out at room temperature in air by exposing enzyme solutions (in Mopso, pH 7.0) containing rose bengal (up to 2 FM) to a 300-W tungsten lamp (15 min in glass tubes at a distance of 25 cm) (18, 25, 26). Enzyme solutions containing the corresponding amounts of rose bengal, but masked with aluminum foil, served as controls. After irradiation, the samples were assayed as described above (in the dark). Protection studies. The various protectants (geranyl pyrophosphate, Mn’*, the linalyl sulfonium analog (4), and inorganic pyrophosphate), alone or in combination, were preincubated with the enzyme (1 ml containing -4 pg protein) for 5 min at 30°C followed by treatment with diethyl pyrocarbonate (or p-hydroxymercuribenzoate in the absence of dithiothreitol(11)) for an additional 15 min at 30°C. Then, [l-3H]geranyl pyrophosphate (and MnCl, if required) was added directly to the treated sample, followed by incubation for an additional 30 min at 30°C to assay for residual cyclase activity. The appropriate adjustment was made for dilution of the labeled substrate. Since the sulfonium ion analog of the linalyl carbonium ion intermediate is itself a potent cyclase inhibitor (11, 22, 27, 28), this material, as well as residual diethyl pyrocarbonate, was removed by gel filtration or dialysis prior to the cyclase assay. This same protocol was employed when testing histidine and cysteine for the ability to protect against inactivation. The appropriate controls without protectant or without inhibitor were included in each experiment. RESULTS

AND DISCUSSION

Inactivation of Limonene Synthase with Diethyl Pyrocarbonate Although there are a few reports (29-31) on the inhibition of monoterpene and sesquiterpene cyclases by the histidine-directed reagent diethyl pyrocarbonate (ethoxyformic anhydride) (12-15), none of these studies have been detailed or have demonstrated specificity (3). Because the glandular trichome secretory cell isolation technique (32,33) has made (-)-(4S)-limonene synthase readily available in microgram quantities at relatively high purity (-40%), comprehensive studies on the inhibition of this cyclase by diethyl pyrocarbonate were undertaken. Preliminary experiments established that the rate of inactivation with diethyl pyrocarbonate (0.1-0.5 InM) increased with pH (in phosphate, Mopso, and Hepes buffers) up to about pH 7.0, consistent with the modification of histidine (imidazole pK, - 6.8) (12). This response was altered by the divalent cations required for cyclase catalysis (downward shift in pH response with Mn2+ (1 mM) and lesser shift with Mg2+ (15 mM)), suggesting an influence of metal ion on histidine exposure or reactivity. Based on these results, most subsequent experiments with diethyl pyrocarbonate were carried out at a pH near neutrality in the absence of divalent metal ion. When limonene synthase was treated at pH 7.0 with a range of diethyl pyrocarbonate levels, a rapid, concentration-dependent loss of cyclase activity was observed (Fig. 1). From a number of experiments of this type, under identical conditions of pH and time, an I50 value of approximately 0.25 mM was consistently determined. Inhibition of cyclase activity was also time-dependent, and the rate of inactivation was pseudo-first-order at concentrations of diethyl pyrocarbonate (0.10,0.25, and 0.50 mM) in great excess of enzyme protein (