Serine Carboxypeptidase-like Acyltransferases Research Articles

Glycosyltransferases from Oat (Avena) Implicated in the Acylation of Avenacins

Plants produce a huge array of specialized metabolites that have important functions in defense against biotic and abiotic stresses. Many of these compounds are glycosylated by family 1 glycosyltransferases (GTs). Oats (Avena spp.) make root-derived antimicrobial triterpenes (avenacins) that provide protection against soil-borne diseases. The ability to synthesize avenacins has evolved since the divergence of oats from other cereals and grasses. The major avenacin, A-1, is acylated with N-methylanthranilic acid. Previously, we have cloned and characterized three genes for avenacin synthesis (for the triterpene synthase SAD1, a triterpene-modifying cytochrome P450 SAD2, and the serine carboxypeptidase-like acyl transferase SAD7), which form part of a biosynthetic gene cluster. Here, we identify a fourth member of this gene cluster encoding a GT belonging to clade L of family 1 (UGT74H5), and show that this enzyme is an N-methylanthranilic acid O-glucosyltransferase implicated in the synthesis of avenacin A-1. Two other closely related family 1 GTs (UGT74H6 and UGT74H7) are also expressed in oat roots. One of these (UGT74H6) is able to glucosylate both N-methylanthranilic acid and benzoic acid, whereas the function of the other (UGT74H7) remains unknown. Our investigations indicate that UGT74H5 is likely to be key for the generation of the activated acyl donor used by SAD7 in the synthesis of the major avenacin, A-1, whereas UGT74H6 may contribute to the synthesis of other forms of avenacin that are acylated with benzoic acid.

Snap-shot of Serine Carboxypeptidase-like Acyltransferase Evolution: The Loss of Conserved Disulphide Bridge is Responsible for the Completion of Neo-functionalization

In this work, it is shown that the At2g23010 gene product encodes 1-O-sinapoyl-β-glucose:1-O-sinapoyl-β-glucose sinapoyltransferase (SST). In contrast to all other functional characterized acyltransferases, the SST protein is highly specific towards this reaction only, and the substrate specificity was correlated to one amino acid substitution. Detailed sequence analysis revealed the lack of the disulphide bond S1 (C78 and C323 in the SMT (sinapoylglucose:malate sinapoyltransferase), that is in SST C80 and D327). The reconstitution of this disulphide bond led to an enzyme accepting many different substrates including disaccharides. Interestingly, the overall changes within the model structures are not very dramatic, but nevertheless, the enzyme models provide some explanations for the broadened substrate specificity: the reconstitution of the disulphide bond provoked more space within the substrate binding pocket simultaneously avoiding electrostatic repulsion. As the SST sequence of A. lyrata also showed the same mutation, the loss of the disulphide bond should has arisen at least 10 mya ago. A Ka/Ks ratio ≤ 1 supports the hypothesis that the loss of this disulphide bond was rather a specification towards a certain reaction than the beginning of a gene death. At the same time, this is also associated with the fixation in the genome.

ChemInform Abstract: Molecular Activities, Biosynthesis and Evolution of Triterpenoid Saponins

AbstractReview: ca. 250 refs.

Stereoselective synthesis of 1- O-β-feruloyl and 1- O-β-sinapoyl glucopyranoses

1- O-β-Feruloyl and 1- O-β-sinapoyl glucopyranoses are two common substrates for serine carboxypeptidase-like acyltransferases and serve as acyl donors in the biosynthesis of numerous secondary metabolites. In addition, they are involved in plant cell wall cross-linking and are also ideal substrates for studying the kinetics of lignification involving hydroxycinnamates. We report the first chemical (and multi-gram scale) synthesis of 1- O-β-feruloyl and 1- O-β-sinapoyl glucopyranoses.

Evolution of serine carboxypeptidase-like acyltransferases in the monocots

The serine carboxypeptidases are a large family of proteases. In higher plants some members of this family have diversified and adopted new functions as acyltransferases required for the synthesis of natural products. We recently reported the first serine carboxypeptidase-like (SCPL) acyltransferase enzyme to be characterised from monocotyledonous plants.1 This enzyme, AsSCPL1, is required for acylation of antimicrobial terpenes (avenacins) that are produced in the roots of oat (Avena spp.) and that provide protection against soil-borne pathogens. The SCPL enzyme family has undergone substantial expansion following the divergence of monocots and dicots. Here we discuss the evolution of the SCPL enzyme family in monocots, their contribution to metabolic diversity, and the roles of these enzymes in biotic and abiotic stress tolerance.

A serine carboxypeptidase-like acyltransferase is required for synthesis of antimicrobial compounds and disease resistance in oats.

Serine carboxypeptidase-like (SCPL) proteins have recently emerged as a new group of plant acyltransferases. These enzymes share homology with peptidases but lack protease activity and instead are able to acylate natural products. Several SCPL acyltransferases have been characterized to date from dicots, including an enzyme required for the synthesis of glucose polyesters that may contribute to insect resistance in wild tomato (Solanum pennellii) and enzymes required for the synthesis of sinapate esters associated with UV protection in Arabidopsis thaliana. In our earlier genetic analysis, we identified the Saponin-deficient 7 (Sad7) locus as being required for the synthesis of antimicrobial triterpene glycosides (avenacins) and for broad-spectrum disease resistance in diploid oat (Avena strigosa). Here, we report on the cloning of Sad7 and show that this gene encodes a functional SCPL acyltransferase, SCPL1, that is able to catalyze the synthesis of both N-methyl anthraniloyl- and benzoyl-derivatized forms of avenacin. Sad7 forms part of an operon-like gene cluster for avenacin synthesis. Oat SCPL1 (SAD7) is the founder member of a subfamily of monocot-specific SCPL proteins that includes predicted proteins from rice (Oryza sativa) and other grasses with potential roles in secondary metabolism and plant defense.

Activities of Arabidopsis sinapoylglucose:malate sinapoyltransferase shed light on functional diversification of serine carboxypeptidase-like acyltransferases

Analysis of the catalytic properties of the serine carboxypeptidase-like (SCPL) 1- O-sinapoyl-β-glucose: l-malate sinapoyltransferase (SMT) from Arabidopsis showed that the enzyme exhibits besides its primary sinapoylation of l-malate, minor hydrolytic and disproportionation activities, producing free sinapic acid and 1,2-di- O-sinapoyl-β-glucose, respectively. The ability of the enzyme to liberate sinapic acid from the donor molecule 1- O-sinapoyl-β-glucose indicates the existence of a short-lived acylenzyme intermediate in the proposed random sequential bi–bi mechanism of catalysis. SMT-catalyzed formation of disinapoylglucose has been corroborated by docking studies with an established homology structure model that illustrates the possible binding of two 1- O-sinapoyl-β-glucose molecules in the active site and the intermolecular reaction of the two glucose esters. The SMT gene is embedded in a tandem cluster of five SCPL sinapoyltransferase genes, which encode enzymes with high amino acid sequence identities and partially overlapping substrate specificities. We assume that in recent duplications of genes encoding SCPL proteins, neofunctionalization of the duplicates to accept 1- O-sinapoyl-β-glucose as acyl donor was gained first, followed by subfunctionalization leading to different acyl acceptor specificities.

Heterologous expression of a serine carboxypeptidase‐like acyltransferase and characterization of the kinetic mechanism

In plant secondary metabolism, beta-acetal ester-dependent acyltransferases, such as the 1-O-sinapoyl-beta-glucose:l-malate sinapoyltransferase (SMT; EC 2.3.1.92), are homologous to serine carboxypeptidases. Mutant analyses and modeling of Arabidopsis SMT (AtSMT) have predicted amino acid residues involved in substrate recognition and catalysis, confirming the main functional elements conserved within the serine carboxypeptidase protein family. However, the functional shift from hydrolytic to acyltransferase activity and structure-function relationship of AtSMT remain obscure. To address these questions, a heterologous expression system for AtSMT has been developed that relies on Saccharomyces cerevisiae and an episomal leu2-d vector. Codon usage adaptation of AtSMT cDNA raised the produced SMT activity by a factor of approximately three. N-terminal fusion to the leader peptide from yeast proteinase A and transfer of this expression cassette to a high copy vector led to further increase in SMT expression by factors of 12 and 42, respectively. Finally, upscaling the biomass production by fermenter cultivation lead to another 90-fold increase, resulting in an overall 3900-fold activity compared to the AtSMT cDNA of plant origin. Detailed kinetic analyses of the recombinant protein indicated a random sequential bi-bi mechanism for the SMT-catalyzed transacylation, in contrast to a double displacement (ping-pong) mechanism, characteristic of serine carboxypeptidases.

Structure determinants and substrate recognition of serine carboxypeptidase-like acyltransferases from plant secondary metabolism

Structures of the serine carboxypeptidase-like enzymes 1- O-sinapoyl-β-glucose: l-malate sinapoyltransferase (SMT) and 1- O-sinapoyl-β-glucose:choline sinapoyltransferase (SCT) were modeled to gain insight into determinants of specificity and substrate recognition. The structures reveal the α/β-hydrolase fold as scaffold for the catalytic triad Ser-His-Asp. The recombinant mutants of SMT Ser173Ala and His411Ala were inactive, whereas Asp358Ala displayed residual activity of 20%. 1- O-sinapoyl-β-glucose recognition is mediated by a network of hydrogen bonds. The glucose moiety is recognized by a hydrogen bond network including Trp71, Asn73, Glu87 and Asp172. The conserved Asp172 at the sequence position preceding the catalytic serine meets sterical requirements for the glucose moiety. The mutant Asn73Ala with a residual activity of 13% underscores the importance of the intact hydrogen bond network. Arg322 is of key importance by hydrogen bonding of 1- O-sinapoyl-β-glucose and l-malate. By conformational change, Arg322 transfers l-malate to a position favoring its activation by His411. Accordingly, the mutant Arg322Glu showed 1% residual activity. Glu215 and Arg219 establish hydrogen bonds with the sinapoyl moiety. The backbone amide hydrogens of Gly75 and Tyr174 were shown to form the oxyanion hole, stabilizing the transition state. SCT reveals also the catalytic triad and a hydrogen bond network for 1- O-sinapoyl-β-glucose recognition, but Glu274, Glu447, Thr445 and Cys281 are crucial for positioning of choline.

Formation of a complex pattern of sinapate esters in Brassica napus seeds, catalyzed by enzymes of a serine carboxypeptidase-like acyltransferase family?

Members of the Brassicaceae accumulate complex patterns of sinapate esters, as shown in this communication with seeds of oilseed rape ( Brassica napus). Fifteen seed constituents were isolated and identified by a combination of high-field NMR spectroscopy and high resolution electrospray ionisation mass spectrometry. These include glucose, gentiobiose and kaempferol glycoside esters as well as sinapine (sinapoylcholine), sinapoylmalate and an unusual cyclic spermidine amide. One of the glucose esters (1,6-di- O-sinapoylglucose), two gentiobiose esters (1- O-caffeoylgentiobiose and 1,2,6′-tri- O-sinapoylgentiobiose) and two kaempferol conjugates [4′-(6- O-sinapoylglucoside)-3,7-di- O-glucoside and 3- O-sophoroside-7- O-(2- O-sinapoylglucoside)] seem to be new plant products. Serine carboxypeptidase-like (SCPL) acyltransferases catalyze the formation of sinapine and sinapoylmalate accepting 1- O-β-acetal esters (1- O-β-glucose esters) as acyl donors. To address the question whether the formation of other components of the complex pattern of the sinapate esters in B. napus seeds is catalyzed via 1- O-sinapoyl-β-glucose, we performed a seed-specific dsRNAi-based suppression of the sinapate glucosyltransferase gene ( BnSGT1) expression. In seeds of BnSGT1-suppressing plants the amount of sinapoylglucose decreased below the HPLC detection limit resulting in turn in the disappearance or marked decrease of all the other sinapate esters, indicating that formation of the complex pattern of these esters in B. napus seeds is dependent on sinapoylglucose. This gives rise to the assumption that enzymes of an SCPL acyltransferase family catalyze the appropriate transfer reactions to synthesize the accumulating esters.

Serine carboxypeptidase-like acyltransferases

In plant secondary metabolism, an alternative pathway of ester formation is facilitated by acyltransferases accepting 1- O-β-acetal esters (1- O-β-glucose esters) as acyl donors instead of coenzyme A thioesters. Molecular data indicate homology of these transferases with hydrolases of the serine carboxypeptidase type defining them as serine carboxypeptidase-like (SCPL) acyltransferases. During evolution, they apparently have been recruited from serine carboxypeptidases and adapted to take over acyl transfer function. SCPL acyltransferases belong to the highly divergent class of α/β hydrolases. These enzymes make use of a catalytic triad formed by a nucleophile, an acid and histidine acting as a charge relay system for the nucleophilic attack on amide or ester bonds. In analogy to SCPL acyltransferases, bacterial thioesterase domains are known which favour transferase activity over hydrolysis. Structure elucidation reveals water exclusion and a distortion of the oxyanion hole responsible for the changed activity. In plants, SCPL proteins form a large family. By sequence comparison, a distinguished number of Arabidopsis SCPL proteins cluster with proven SCPL acyltransferases. This indicates the occurrence of a large number of SCPL proteins co-opted to catalyse acyltransfer reactions. SCPL acyltransferases are ideal systems to investigate principles of functional adaptation and molecular evolution of plant genes.

Biochemical Characterization of Sinapoylglucose:Choline Sinapoyltransferase, a Serine Carboxypeptidase-like Protein That Functions as an Acyltransferase in Plant Secondary Metabolism

Recently, serine carboxypeptidase-like (SCPL) proteins that catalyze transacylation reactions in plant secondary metabolism have been identified from wild tomato and Arabidopsis. These include sinapoylglucose: choline sinapoyltransferase (SCT), an enzyme that functions in Arabidopsis sinapate ester synthesis. SCT and the other known SCPL acyltransferases all share the conserved serine, aspartic acid, and histidine residues employed for catalysis by classical serine carboxypeptidases, although the importance of these residues and the mechanism by which this class of SCPL proteins catalyze acyltransferase reactions is unknown. To characterize further SCT and its catalytic mechanism, we have employed the Saccharomyces cerevisiae vacuolar protein localization 1 mutant, which secretes the serine carboxypeptidase, carboxypeptidase Y, and other proteins normally targeted to the vacuole. When expressed in this strain, SCT is similarly secreted. SCT has been purified from the yeast medium and used for kinetic characterization of the protein. Immunological analysis of SCT has revealed that the expected 50-kDa mature protein is proteolytically processed in yeast and in planta, most likely resulting in the production of a heterodimer derived from a 30- and 17-kDa polypeptide.

The sng2 mutant of Arabidopsis is defective in the gene encoding the serine carboxypeptidase-like protein sinapoylglucose:choline sinapoyltransferase.

Serine carboxypeptidase-like (SCPL) proteins have traditionally been assigned roles in the hydrolytic processing of proteins; however, several SCPL proteins have recently been identified as catalysts in transacylation reactions of plant secondary metabolism. The novel functions of these enzymes suggest a catalytic diversity for plant SCPL proteins that extends beyond simple hydrolysis reactions. Characterization of the Arabidopsis sng2 (sinapoylglucose accumulator 2) mutant has identified another SCPL protein involved in plant secondary metabolism. The sng2 mutant was isolated by screening seed extracts for altered levels of sinapate esters, a group of phenylpropanoid compounds found in Arabidopsis and some other members of the Brassicaceae. Homozygous sng2 seeds accumulate sinapoylglucose instead of sinapoylcholine, and have increased levels of choline and decreased activity of the enzyme sinapoylglucose:choline sinapoyltransferase (SCT). Cloning of the SNG2 gene by a combination of map-based and candidate gene approaches demonstrates that SCT is another member of the growing class of SCPL acyltransferases involved in plant secondary metabolism.