Structure-function relationships of SCPL acyltransferases

CARSTEN MILKOWSKI
Leibniz Institute of Plant Biochemistry
Dep. Secondary Metabolism
Weinberg 3
D-06120 Halle (Saale)
Carsten.Milkowski@ipb-halle.de
http://www.ipb-halle.de/en/research/secondary-metabolism/

DIETER STRACK
Leibniz Institute of Plant Biochemistry
Dep. Secondary Metabolism
Weinberg 3
D-06120 Halle (Saale)
Dieter.Strack@ipb-halle.de
http://www.ipb-halle.de/en/research/secondary-metabolism/

MILTON T. STUBBS
Department of Physical Biotechnology
Martin Luther University
Kurt-Mothes-Str. 3
D-06120 Halle (Saale)
stubbs@biochemtech.uni-halle.de
http://www.biochemtech.uni-halle.de/biotechnologie/xray/

References
Milkowski C., Strack D (2004) Serine carboxypeptidase-like acyltransferases. Phytochemistry 65: 517-524 (Review)
In plant secondary metabolism, an alternative pathway of ester formation is facilitated by acyltransferases accepting 1-O-ß-acetal esters (1-O-ß-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 a/ß 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.

Baumert A, Milkowski C, Schmidt J, Nimtz M, Wray V, Strack D (2005) Formation of a complex pattern of sinapate esters in Brassica napus seeds, catalysed by enzymes of a serine carboxypeptidase-like acyltransferase family. Phytochemistry 66:1334-1345.
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-ß-acetal esters (1-O-ß-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-ß-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.

Stehle F, Brandt W, Milkowski C, Strack D (2006) Structure determinants and substrate recognition of serine carboxypeptidase-like acyltransferases from plant secondary metabolism. FEBS Lett. 580: 6366-74.
Structures of the serine carboxypeptidase-like enzymes 1-O-sinapoyl-ß-glucose:L-malate sinapoyltransferase (SMT) and 1-O-sinapoyl-ß-glucose:choline sinapoyltransferase (SCT) were modeled to gain insight into determinants of specificity and substrate recognition. The structures reveal the a/ß 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-ß-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-ß-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-ß-glucose recognition, but Glu274, Glu447, Thr445 and Cys281 are crucial for
positioning of choline.

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