IN THIS ISSUE

Acyltransferases in Protease's Clothing

One of the most intriguing questions in plant secondary metabolism concerns the evolution of the genes required for the rapid diversification of plant chemistry. In particular, the mechanisms are beginning to be understood by which evolution recruits primary and intermediary metabolism genes to carry out novel reactions with existing substrates to create the extraordinary diversity that characterizes the chemistry of higher plants. One mechanism that has been recently studied involves the activation and transacylation of phenolic and fatty acids. Although this reac-tion is frequently assumed to proceed via thioester-dependent mechanisms (CoA), alternative CoA-independent activation mechanisms are a central feature of plant secondary metabolism. These involve the UDPglucose-dependent transglucosylation of phenolic and fatty acids to yield 1-O-ß-acyl acetals that most frequently occur as the 1-O-ß-ester of glucose. Such activated es-ters exhibit group transfer potentials (-35.7 kJ mol^-1) similar to those of thioesters (Mock and Strack 1993 ). Their role in secondary metabolism was first implicated in 1972 by Kojima and Uritani, in the biosynthesis of chlorogenic acid (caffeoylquinic acid), a compound for which both CoA-dependent and 1-O-ß-caffeoylglucose–dependent pathways are now known. In the former route, caffeoyl-CoA is the donor for transfer to quinic acid. In the latter route, a 1-O-ß-caffeoylglucose:quinic acid acyltransferase utilizes the group transfer potential of 1-O-ß-caffeoylglucose to transesterify quinic acid.

Although chlorogenic acid is one of the most abundant and widespread phenolic constituents of plants, the mechanism of transacylation from 1-O-ß-caffeoylglucose to quinic acid is not known. In addition, a remarkable number of other plant metabolites are also known to be derived through transesterification from an activated 1-O-ß-acyl acetal. Among others, these metabolites include the myo-inositol-based ester conjugates of indole acetic acid; gallotannins (gallic acid esters of glucose characteristic of oak and sumac species); feruloylated betacyanins; sinapate esters of glucose, choline, and malate characteristic of Brassica species; hydroxycinnamoyl esters of anthocyanins; isochlorogenic acid (di-O-caffeoylquinic acid), caffeoylglucaric acid, and caffeoylgalactonic acid; and the glucose and sucrose fatty acyl esters of trichome secretions from some solanaceous species. In each of these cases, the donor molecule responsible for transacylation is a 1-O-ß-acyl acetal, and in many cases, both the biochemistry and the genes underlying the UDPG-dependent activation of the acid are well characterized. However, despite the importance of IAA, phenolic, and fatty acid metabolism to plant biochemistry, the nature and regulation of the enzymes catalyzing the transesterification of 1-O-ß-acetal esters has remained unknown. Two recent papers not only shed considerable light on the mechanism of acyl transfer from 1-O-ß-acetal esters, but also reveal how acyltransferases of secondary metabolism have evolved from serine proteinases.

Writing in the June 6 issue of PNAS, Li and Steffens 2000 report the isolation of a cDNA encoding the acyltransferase responsible for the formation of diacyl glucose, an intermediate in the biosynthesis of glucose polyes-ters. These compounds, typically composed of glucose or sucrose esterified to 3 or more fatty acids, are secreted from glandular trichomes and constitute part of the insect defensive chemistry of wild Solanaceae such as Lycopersicon pennellii and Solanum berthaultii. In this case, a 1-O-ß-isobutyryl glucose donor is utilized in the transfer of the isobutyryl moiety to another 1-O-ß-isobutyryl glucose molecule, forming 1,2-di-O-diisobutyryl-ß-glucose (Ghangas and Steffens 1993 , Ghangas and Steffens 1995 ). Working from an amino acid sequence derived from the acyltransferase purified from L. pennellii glandular trichomes, the acyltransferase cDNA was found to encode a protein with significant similarity to serine carboxypeptidases. From the point of view of the known functions of serine carboxypeptidases—which are exclusively hydrolytic—identification of the L. pennellii acyltransferase as a serine carboxypeptidase-like protein was a surprise.

As a class, serine proteinases, of which yeast carboxypeptidase Y is perhaps one of the most well-studied, form a functionally diverse group of enzymes. By definition, serine carboxypeptidases possess a characteristic catalytic triad usually composed of Ser-His-Asp. The Ser residue is unusually reactive because of its interaction with the His imidazole nitrogen. In the catalytic mechanism of serine carboxypeptidases, this Ser undergoes acylation as a consequence of its nucleophilic attack on the carbonyl carbon of a peptide substrate, forming a tetrahedral acyl-enzyme intermediate. This intermediate is then decomposed by water, liberating the bound peptide and regenerating the reactive Ser. As a result of convergent molecular evolution, the Ser-His-Asp catalytic triad is also responsible for the activities of endopeptidases such as trypsin and subtilisin. Serine carboxypeptidases are primarily known for catabolic or processing functions, for example, protein turnover or modification of precursor proteins, mobilization of seed storage protein reserves, etc. However, recruitment of active serine motifs for other hydrolytic functions is widespread (Dodson and Wlodawer 1998 ). For example, some lipases and esterases possess active Ser-based catalytic triad centers. A cyanogenic hydroxynitrile lyase from sorghum and a fungal cutinase also share significant sequence similarity to serine carboxypeptidases, and both functionally employ an active Ser-based catalytic triad in catalysis.

Moreover, many serine carboxypeptidases also exhibit esterase activity on selected substrates, as expected from the chemical similarity of ester and amide bonds. However, transesterification, as that exemplified by the L. pennellii acyltranferase, is known only in serine carboxypeptidase reactions performed in anhydrous organic solvents (Riva et al. 1998 ), or at extremes of alkaline pH (Widmer and Johansen 1979 ). Therefore, the reaction catalyzed by the L. pennellii acyltransferase appears quite unique for an enzyme most closely resembling a protease. However, the reaction is regiospecific, forming exclusively a 1,2-di-O-acyl glucose product. In addition, the acyltransferase exhibits no carboxypeptidase activity toward any model substrate tested. Most importantly, the acyltransferase can be shown directly to rely upon an active Ser residue for catalysis. Diisopropylfluorophosphate (DFP) is an irreversible inhibitor of active Ser-based enzymes, and was developed as a chemical warfare agent targeted against acetylcholinesterases. DFP, which reacts irreversibly with active Ser residues to form a covalent DFP-enzyme complex, completely inhibits L. pennellii acyltransferase activity, and ¹⁴C-DFP covalently modifies the subunit predicted to carry the active Ser residue. Therefore, the active Ser plays a critical function in catalysis of acyl transfer from 1-O-acyl-ß-glucose, and confirms that the glucose acyltransferase utilizes a serine carboxypeptidase-like catalytic triad to accomplish the synthesis of glucose polyesters. By analogy to serine proteases, the mechanism of acyl transfer from 1-O-ß-acyl acetals involves nucleophilic attack of the active Ser hydroxyl on the ester carbonyl carbon of 1-O-ß-acylglucose to form a tetrahedral enzyme-bound acyl intermediate, releasing glucose. This transition state would then be subject to attack by the 2-hydroxyl of a second 1-O-ß-acylglucose, resulting in formation of 1,2-di-O-ß-acylglucose, and regenerating the active Ser.

Following the initial identification of the L. pennelllii glucose acyltransferase as a SCPL protein, four other serine proteinase-based acyltransferases in sugar polyester biosynthesis have been identified. Three of these (accessions AF006078, AF006079, AF006080) catalyze isobutyryl transfer to glucose and also are derived from the serine carboxypeptidase family that includes yeast carboxypeptidase Y. The remaining gene, an unpublished isobutyrylglucose:sucrose isobutyryltransferase identified by Nancy Eannetta at Cornell, is a monomeric 70-kD protein in the serine proteinase family of which subtilisin is the most well-known member. The sucrose acyltransferase activity of this gene's product is also abolished by treatment with DFP, again confirming the centrality of the catalytic triad in transacylation by this protein.

On pages 1295–1306 of this issue, Lehfeldt et al. identify a serine carboxypeptidase-like protein as an acyltransferase involved in the biosynthesis of sinapoyl malate, an abundant phenolic ester of Arabidopsis and its relatives. The sinapate esters play a role in UV protection in Arabidopsis, and both their UV fluorescence and in vivo dispensibility make them attractive tar-gets for genetic analysis (Lorenzen et al. 1996 ). In Arabidopsis, ß-1-O-sinapoylglucose is the activated donor of sinapate for biosynthesis of both sinapoyl malate and sinapoyl choline, which accumulate in leaves and seeds, respectively (Strack 1982 ). Mutations at sng1 (sinapoylglucose accumulator 1) accumulate sinapoylglucose instead of sinapoyl malate in leaves and lack detectable sinapoylglucose:malate sinalpoyltransferase activity (SMT). This work, a collaboration between two labs involved in the genetics (Chapple) and biochemistry (Strack) of sinapate metabolism confirms a specific prediction of the PNAS paper, namely that the transacylation reaction responsible for the formation of sinapoylmalate from ß-1-O-sinapoylglucose and malate, is catalyzed by a serine carboxypeptidase-like protein. Lehfeldt et al. not only complemented sng1 with a genomic SNG1 clone, restoring accumulation of sinapoylmalate, but also expressed an SNG1 cDNA in Escherichia coli and demonstrated synthesis of sinapoylmalate from ß-1-O-sinapoylglucose and malate. The genetic approach used by Lehfeldt et al. to identify the sinapoyl transacylase as a serine carboxypeptidase-like protein removes any remaining question that these proteins act as acyltransferases in vivo. Interestingly, both previous biochemical research (Strack and Sharma 1985 ) and sorting algorithms predict the vacuolar location of SMT, similar to many carboxypeptidases.

The evolution of serine carboxypeptidases to active-serine-based acyltransferases—essentially the change from a hydrolytic to a transacylation function—requires that water be excluded from the active site once the acyl-enzyme intermediate is formed, or that the enzyme activate the hydroxyl of the acceptor (e.g., malate) sufficiently to promote the reaction leading to transacylation over that of hydrolysis. At present, the sequences of known active-serine-based acyltransferases do not disclose features that permit assignment of a proteolytic or transacylating function. Even if this were possible, the diversity of existing secondary metabolites suggests that one is still left with the problem of identifying the substrates utilized by each putative active serine-based acyltransferase.

Indeed, on the bacterial artificial chromosome demonstrated by Lefeldt et al. as bearing sinapoyl glucose: malate sinapoylglucose acyltransferase, four additional genes encoding serine carboxypeptidase-like proteins were also identified, leading to the possibility that clustering of the sinapate ester biosynthetic genes had occurred during evolution of this pathway. However, none of the fast neutron–induced deletion mutants that disrupt these genes led to defects in the steady state accumulation of products of sinapate ester biosynthesis. Therefore, it is likely that sinapoyl glucose:choline sinapoylglucose acyltransferase resides elsewhere in the genome. This raises interesting questions about the biochemical roles of the additional serine carboxypeptidase-like proteins, and the approaches to be taken to identify their function.

As pointed out by both Li and Steffens, and Lehfeldt et al., the presence of around 40 Arabidopsis genes encoding serine carboxypeptidase-like proteins implies the existence of a larger family of these genes, perhaps numbering 60, whose function remains to be shown. Similarly, in tomato, both the serine carboxypeptidase and subtilisin-like proteinase families are large and complex. Clearly, some of these genes provisionally annotated as proteinases will prove to be bona fide proteases involved in mobilization of storage proteins, protein maturation, etc. However, these studies imply that an entire family of genes with many novel transacylase activities has now been identified. If one considers further that the catalytic triad in other members of the serine proteinase superfamily could be adapted for acyltransferase function—as suggested by the identification of a subtilisin-like sucrose acyltransferase—the numbers of such genes could be very high. Similarly, one can imagine mechanisms by which any of the other proteinase families could evolve to carry out transacylations or by which proteinases could evolve to carry out phosphoryl transfer from ß-acetal ester intermediates such as glucose-1-phosphate. These possibilities pose a formidable challenge for both database annotation and functional genomics. Clearly, the work reported by Lehfeldt et al. in this issue of THE PLANT CELL broadens the known functions of serine proteinases from primarily hydrolytic functions to participants in the biosynthesis of a broad array of metabolites and provides a dramatic illustration of the process by which the recruitment of primary reactions leads to the diversity of plant secondary metabolites.

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Cloning of the SNG1 Gene of Arabidopsis Reveals a Role for a Serine Carboxypeptidase-like Protein as an Acyltransferase in Secondary Metabolism

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