Role of a GDSL lipase‐like protein as sinapine esterase in Brassicaceae

Abstract

SummaryThe seeds of most members of the Brassicaceae accumulate high amounts of sinapine (sinapoylcholine) that is rapidly hydrolyzed during early stages of seed germination. One of three isoforms of sinapine esterase activity (BnSCE3) has been isolated from Brassica napus seedlings and subjected to trypsin digestion and spectrometric sequencing. The peptide sequences were used to isolate BnSCE3 cDNA, which was shown to contain an open reading frame of 1170 bp encoding a protein of 389 amino acids, including a leader peptide of 25 amino acids. Sequence comparison identified the protein as the recently cloned BnLIP2, i.e. a GDSL lipaselike protein, which displays high sequence identity to a large number of corresponding plant proteins, including four related Arabidopsis lipases. The enzymes belong to the SGNH protein family, which use a catalytic triad of Ser-Asp-His, with serine as the nucleophile of the GDSL motif. The corresponding B. napus and Arabidopsis genes were heterologously expressed in Nicotiana benthamiana leaves and proved to confer sinapine esterase activity. In addition to sinapine esterase activity, the native B. napus protein (BnSCE3/ BnLIP2) showed broad substrate specificity towards various other choline esters, including phosphatidylcholine. This exceptionally broad substrate specificity, which is common to a large number of other GDSL lipases in plants, hampers their functional analysis. However, the data presented here indicate a role for the GDSL lipase-like BnSCE3/BnLIP2 as a sinapine esterase in members of the Brassicaceae, catalyzing hydrolysis of sinapine during seed germination, leading, via 1-O-sinapoyl-b-glucose, to sinapoyl-L-malate in the seedlings.

The Phenylpropanoid Pathway in Arabidopsis

“…Hydrolysis of the compound in germinating seedlings by sinapoylcholine esterase (SCE) releases choline for phospholipid synthesis and sinapate which is re-esterified by SGT to form sinapoylglucose (Tzagoloff, 1963;Nurmann and Strack, 1979;Strack et al, 1980;Clauß et al, 2008). There are four Arabidopsis genes whose sequences are similar to the gene encoding SCE in B. napus (Clauß et al, 2008).…”

“…The proteins encoded by these three genes exhibit SCE activity and are highly expressed in 3-day-old seedlings (Table 1). One of these genes (At1g28670) is also expressed in adult leaves, suggesting a possible alternative role in mature plants (Clauß et al, 2008). Interestingly, the three Arabidopsis SCEs are not SCPL proteins like SMT and SCT, but instead are similar in sequence to GDSL serine lipases/esterases, hydrolytic enzymes with broad substrate specificities (Akoh et al, 2004).…”

“…Interestingly, the three Arabidopsis SCEs are not SCPL proteins like SMT and SCT, but instead are similar in sequence to GDSL serine lipases/esterases, hydrolytic enzymes with broad substrate specificities (Akoh et al, 2004). The Arabidopsis SCEs appear to have the conserved Ser, Gly, Asn, and His catalytic residues characteristic of GDSL proteins, and may employ a Ser-Asp-His catalytic triad similar to the ones found in SCPL proteins (Clauß et al, 2008). Also like SCPL proteins, the Arabidopsis SCEs may be the evolutionary result of enzymes involved in primary plant metabolism being "recruited" for roles in secondary metabolism (Clauß et al, 2008).…”

Identification and Determination of Phenolic Compounds in Rapeseed Meals (Brassica napus L.)

“…On the other hand, in the Extraction II, rapeseed meal was extracted with adding of 10% methanol contained 0.1% phosphoric acid together with TBHQ. And hydrochloric acid was then added with different ratios (1:1, 1:2, 1:3, v/v; labeled as EII-A, EII-B, EII-C, respectively) [12]. After each extraction process, the mixtures were centrifuged at 12,000 rpm at 4˚C for 15 min.…”

Requirement of Catalytic-Triad and Related Amino Acids for the Acyltransferase Activity ofTanacetum cinerariifoliumGDSL Lipase/Esterase TcGLIP for Ester-Bond Formation in Pyrethrin Biosynthesis

“…1A), chrysanthemoyl CoA and (S)-pyrethrolone were added to a crude enzyme from the flower bud, resulting in the production of pyrethrin I. Purifying this enzyme and cloning the gene unexpectedly led to the discovery that the isolated enzyme showed similarities to GDSL lipases/esterases which have been named after the Gly-Asp-Ser-Leu motif. 4) This family consists of many members with such diverse functions as ceramidase, 5) sinapine esterase, 6) transesterification, 7) cutin synthesis 8) and cutinase activities 9) in plants. 10) We confirmed that the recombinant enzyme expressed in Escherichia coli as a fusion with maltosebinding protein (MBP) showed acyltransferase activity for pyrethrin synthesis.…”