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

Abstract

Structures of the serine carboxypeptidase-like enzymes 1-O-sinapoyl-b-glucose:L L-malate sinapoyltransferase (SMT) and 1-O-sinapoyl-b-glucose:choline sinapoyltransferase (SCT) were modeled to gain insight into determinants of specificity and substrate recognition. The structures reveal the a/bhydrolase 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-b-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-Osinapoyl-b-glucose and L L-malate. By conformational change, Arg322 transfers L 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-b-glucose recognition, but Glu274, Glu447, Thr445 and Cys281 are crucial for positioning of choline.

The Phenylpropanoid Pathway in Arabidopsis

“…Based on these results, and a combination of mutagenesis experiments and molecular modeling, Stehle and coworkers suggest that SMT may employ a random sequential bi-bi mechanism of catalysis (Stehle et al, 2006;Stehle et al, 2008a;Stehle et al, 2009). …”

“…similar, functioning as acyltransferases that share the same activated donor molecule (sinapoylglucose) but whose acyl acceptor substrates differ (Fraser et al, 2007). Although a structure of an Arabidopsis SCPL sinapoylglucose acyltransferase has yet to be produced, modeling and experimental data have identified a number of candidate amino acid residues that may be involved in protein-substrate interactions, and suggest that SMT, SCT and the other Arabidopsis SCPL sinapoylglucose acyltransferases have evolved from a common hydrolytic ancestor (Lehfeldt et al, 2000;Shirley et al, 2001;Shirley and Chapple, 2003;Fraser et al, 2005;Stehle et al, 2006;Fraser et al, 2007;Stehle et al, 2008a;Stehle et al, 2009). Research on the other 46 Arabidopsis SCPL proteins is likely to shed further light on the role of enzymatic evolution in plant secondary metabolism, both in Arabidopsis and higher plants in general.…”

A Serine Carboxypeptidase-Like Acyltransferase Is Required for Synthesis of Antimicrobial Compounds and Disease Resistance in Oats

“…However, not all SCPL proteins undergo such processing (Hause et al, 2002;Stehle et al, 2006). Comparison of the amino acid sequences of oat SCPL1 with those of characterized SCP and SCPL proteins (some of which undergo cleavage and some of which do not) reveals that SCPL1 has an extended linker region typical of SCPL proteins that are cleaved into two subunits ( Figure 7A).…”

Evolution of serine carboxypeptidase-like acyltransferases in the monocots

“…1,[3][4][5][6][7][8][9][10][11][12][13][14][15][16] Previously characterized SCPL acyltransferases from dicots include enzymes required for synthesis of glucose polyesters implicated in insect defence in tomato 3 and for synthesis of UV-protectant sinapate esters in Arabidopsis. 4 Our finding that the oat AsSCPL1 enzyme acylates antifungal triterpenes that are required for disease resistance extends the known range of functions of this family, and also suggests that the SCPL acyltransferase family originated before the divergence of the monocots and dicots.…”

“…1,[3][4][5][6][7][8][9][10][11][12][13][14][15][16] To investigate the possible functions of other monocot members of this clade we analysed the expression pattern of the gene encoding the closest relative to AsSCPL1 in rice (Os10g01134) surrounding the catalytic serine residue, which is conserved across all members of Clade IA (the acyltransferase clade), is not found in P. patens or C. reinhardtii SCPL proteins. This motif is likely to be important for formation of a hydrogen bonding network that allows binding of the glucose ester substrates.…”