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Publications - Cell and Metabolic Biology

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Publications

Stehle, F.; Brandt, W.; Stubbs, M. T.; Milkowski, C.; Strack, D.; Sinapoyltransferases in the light of molecular evolution Phytochemistry 70, 1652-1662, (2009) DOI: 10.1016/j.phytochem.2009.07.023

Acylation is a prevalent chemical modification that to a significant extent accounts for the tremendous diversity of plant metabolites. To catalyze acyl transfer reactions, higher plants have evolved acyltransferases that accept β-acetal esters, typically 1-O-glucose esters, as an alternative to the ubiquitously occurring CoA-thioester-dependent enzymes. Shared homology indicates that the β-acetal ester-dependent acyltransferases are derived from a common hydrolytic ancestor of the Serine CarboxyPeptidase (SCP) type, giving rise to the name Serine CarboxyPeptidase-Like (SCPL) acyltransferases. We have analyzed structure–function relationships, reaction mechanism and sequence evolution of Arabidopsis 1-O-sinapoyl-β-glucose:l-malate sinapoyltransferase (AtSMT) and related enzymes to investigate molecular changes required to impart acyltransferase activity to hydrolytic enzymes. AtSMT has maintained the catalytic triad of the hydrolytic ancestor as well as part of the H-bond network for substrate recognition to bind the acyl acceptor l-malate. A Glu/Asp substitution at the amino acid position preceding the catalytic Ser supports binding of the acyl donor 1-O-sinapoyl-β-glucose and was found highly conserved among SCPL acyltransferases. The AtSMT-catalyzed acyl transfer reaction follows a random sequential bi-bi mechanism that requires both substrates 1-O-sinapoyl-β-glucose and l-malate bound in an enzyme donor–acceptor complex to initiate acyl transfer. Together with the strong fixation of the acyl acceptor l-malate, the acquisition of this reaction mechanism favours transacylation over hydrolysis in AtSMT catalysis. The model structure and enzymatic side activities reveal that the AtSMT-mediated acyl transfer proceeds via a short-lived acyl enzyme complex. With regard to evolution, the SCPL acyltransferase clade most likely represents a recent development. The encoding genes are organized in a tandem-arranged cluster with partly overlapping functions. With other enzymes encoded by the respective gene cluster on Arabidopsis chromosome 2, AtSMT shares the enzymatic side activity to disproportionate 1-O-sinapoyl-β-glucoses to produce 1,2-di-O-sinapoyl-β-glucose. In the absence of the acyl acceptor l-malate, a residual esterase activity became obvious as a remnant of the hydrolytic ancestor. With regard to the evolution of Arabidopsis SCPL acyltransferases, our results suggest early neofunctionalization of the hydrolytic ancestor toward acyltransferase activity and acyl donor specificity for 1-O-sinapoyl-β-glucose followed by subfunctionalization to recognize different acyl acceptors.
Publications

Stehle, F.; Stubbs, M. T.; Strack, D.; Milkowski, C.; Heterologous expression of a serine carboxypeptidase-like acyltransferase and characterization of the kinetic mechanism FEBS J. 275, 775-787, (2008) DOI: 10.1111/j.1742-4658.2007.06244.x

In plant secondary metabolism, β‐acetal ester‐dependent acyltransferases, such as the 1‐O ‐sinapoyl‐β‐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.
Publications

Stehle, F.; Brandt, W.; Milkowski, C.; Strack, D.; Structure determinants and substrate recognition of serine carboxypeptidase-like acyltransferases from plant secondary metabolism FEBS Lett. 580, 6366-6374, (2006) DOI: 10.1016/j.febslet.2006.10.046

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.
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