Literature summary for 4.2.99.23 extracted from

Nomura, T.
Function and application of a non-ester-hydrolyzing carboxylesterase discovered in tulip (2017), Biosci. Biotechnol. Biochem., 81, 81-94 .

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Application

Application	Comment	Organism
synthesis	PaA and PaB are considered to be useful natural products with potential applications as synthetic intermediates in the production of several bioactive compounds, antimutagenic agents, insect repellents, and as monomers of functional biobased polymers, application of TCE to produce PaB, overview	Tulipa gesneriana

Cloned(Commentary)

Cloned (Comment)	Organism
gene TgTCEB1, sequence comparisons and unrooted phylogenetic tree, functional recombinant expression of the mature dimeric enzyme in Escherichia coli. Although the recombinant enzyme is functionally expressed as N-terminal His-tagged protein, removal of the vector-derived sequence including His-tag by protease treatment almost doubles its activity, allowing for accurate assessment of enzyme characteristics. Recombinant transient expression of GFP-tagged N-terminal transit peptides of TCEB in onion epidermal cell plastids	Tulipa gesneriana

Cloned (Comment)

Organism

gene TgTCEB1, sequence comparisons and unrooted phylogenetic tree, functional recombinant expression of the mature dimeric enzyme in Escherichia coli. Although the recombinant enzyme is functionally expressed as N-terminal His-tagged protein, removal of the vector-derived sequence including His-tag by protease treatment almost doubles its activity, allowing for accurate assessment of enzyme characteristics. Recombinant transient expression of GFP-tagged N-terminal transit peptides of TCEB in onion epidermal cell plastids

Tulipa gesneriana

Protein Variants

Protein Variants	Comment	Organism
additional information	enzyme immobilization of TCEB for improved synthesis of PaB, overview	Tulipa gesneriana

Inhibitors

Inhibitors	Comment	Organism	Structure
phenylmethylsulfonyl fluoride	remarkably reduces the activity of the native enzyme purified from tulip	Tulipa gesneriana

Localization

Localization	Comment	Organism	GeneOntology No.	Textmining
chloroplast	the enzyme's transit peptide functions as a plastid-sorting signal for mature TCE polypeptides	Tulipa gesneriana	9507	-

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.
6-tuliposide A	Tulipa gesneriana	very low activity	tulipalin A + D-glucose	-	?
6-tuliposide B	Tulipa gesneriana	-	tulipalin B + D-glucose	-	?
additional information	Tulipa gesneriana	TCE enzymes are lactone-forming enzymes and a non-ester-hydrolyzing carboxylesterase, and specifically catalyze intramolecular transesterification, but not hydrolysis	?	-	?

Organism

Organism	UniProt	Comment	Textmining
Tulipa gesneriana	-	-	-

Posttranslational Modification

Posttranslational Modification	Comment	Organism
proteolytic modification	comparison of the full-length TgTCE polypeptides with the N-termini of the corresponding native TCEs purified from tulip tissues indicated that the enzymes are translated as precursor polypeptides before being processed to mature polypeptides via cleavage of the N-terminal transit peptides	Tulipa gesneriana

Purification (Commentary)

Purification (Comment)	Organism
native enzyme from pollen grains	Tulipa gesneriana

Reaction

Reaction	Comment	Organism	Reaction ID
6-tuliposide B = tulipalin B + D-glucose	putative reaction mechanism of TCE enzymes, overview. The reaction begins with a nucleophilic attack by the catalytic Ser, whose hydroxyl group is activated by the charge relay of the catalytic triad, on the carbonyl carbon of 6-Pos. This is followed by the formation of a tetrahedral intermediate, which is stabilized by the oxyanion hole structure contributed by the two Gly residues of the HGG motif. Then, following the elimination of glucose, the acyl-enzyme complex is formed, and an intramolecular nucleophilic attack by a terminal hydroxyl group of 6-Pos, but not by water, occurs. This nucleophilic attack results in the formation of the five-membered ring structure of Pa. If the acylenzyme complex is subjected to a nucleophilic attack by activated water, then ?-hydroxy acids are released as hydrolytic products, but this process never occurs in the TCE reactions	Tulipa gesneriana	a

Source Tissue

Source Tissue	Comment	Organism	Textmining
additional information	constitutive enzyme expression. The TCEs and 6-Pos are well separated by spatial compartmentation in intact tulip cells	Tulipa gesneriana	-
pollen	-	Tulipa gesneriana	-

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
6-tuliposide A	very low activity	Tulipa gesneriana	tulipalin A + D-glucose	-	?
6-tuliposide A	i.e. 6-PosA, enzyme TCEB shows 0.4-2.0% activity with 6-PosA compared to 6-PosB	Tulipa gesneriana	tulipalin A + D-glucose	-	?
6-tuliposide B	-	Tulipa gesneriana	tulipalin B + D-glucose	-	?
6-tuliposide B	i.e. 6-PosB, preferred substrate	Tulipa gesneriana	tulipalin B + D-glucose	-	?
additional information	TCE enzymes are lactone-forming enzymes and a non-ester-hydrolyzing carboxylesterase, and specifically catalyze intramolecular transesterification, but not hydrolysis	Tulipa gesneriana	?	-	?
additional information	enzme TCEB preferentially catalyzes the conversion of 6-PosB to PaB, and catalyzes intramolecular transesterification of 6-tuliposide B (6-PosB) to form tulipalins B (PaB), but neither the hydrolysis of 6-Pos nor Pa. 1alpha- and 1beta-anomers exist for 6-PosB, and both anomers serve as substrates for TCEB. All three TCEB isoforms preferentially catalyze the conversion of 6-PosB to PaB, with about 0.4-2% activities toward 6-PosA	Tulipa gesneriana	?	-	?

Subunits

Subunits	Comment	Organism
homodimer	-	Tulipa gesneriana

Synonyms

Synonyms	Comment	Organism
6-PosB-converting enzyme	-	Tulipa gesneriana
ignition enzyme	-	Tulipa gesneriana
non-ester-hydrolyzing TCE	-	Tulipa gesneriana
PosB-converting enzyme	-	Tulipa gesneriana
TCE	-	Tulipa gesneriana
TCEB	-	Tulipa gesneriana
TCEB1	-	Tulipa gesneriana
tuliposide-converting enzyme	-	Tulipa gesneriana

General Information

General Information	Comment	Organism
evolution	TCE enzymes belong to the carboxylesterase family in the alpha/beta-hydrolase fold superfamily, and specifically catalyze intramolecular transesterification, but not hydrolysis, functional diversity of carboxylesterases and related alpha/beta-hydrolase enzymes and proteins in plants, overview. TgTCEA1 and TgTCEB1 polypeptides show 52% identity to each other and approximately 40-45% identities to the sequences annotated as plant carboxylesterases. TgTCEs catalyze only intramolecular transesterification, while other plant carboxylesterases catalyze the hydrolysis of carboxylic esters to form carboxylic acids and alcohols. TgTCE polypeptides have sequence motifs typical of class I carboxylesterases in the alpha/beta-hydrolase fold superfamily, including the HGG motif. The two Gly residues in this motif are involved in the formation of an oxyanion hole structure during the formation of the acyl-enzyme complex. The other conserved motif is the catalytic triad Ser, Asp, and His, of which the catalytic Ser is present within the conserved pentapeptide sequence Gly-X-Ser-X-Gly	Tulipa gesneriana
physiological function	tuliposides (Pos) and tulipalins (Pa) are the major defensive secondary metabolites in tulip (Tulipa gesneriana). So far, seven analogues of Pos have been reported: 1-PosA, 1-PosB, 6-PosA, 6-PosB, PosD, PosE, and PosF, which differ in the number, position, and structure of the acyl side chain. They are categorized into monoacyl-glucose type (1-Pos and 6-Pos) and 1,6-diacyl-glucose type (PosD, PosE, and PosF), overview. The tuliposide-converting enzyme (TCE), a non-ester-hydrolyzing carboxylesterase, catalyzes the conversion of major defensive secondary metabolites, tuliposides, to antimicrobial tulipalins. PaA has been shown to have potent insecticidal activity against several insect pests, such as Thrips palmi. PaA functions as an antifungal agent rather than an antibacterial agent, and vice versa for PaB. Enzyme-mediated activation of postinhibitins	Tulipa gesneriana