Thioesterase from Passion Fruit
J. Agric. Food Chem., Vol. 56, No. 15, 2008 6629
was then treated with a trifunctional fluorophosphonate activity
probe (TriFPP). The custom-synthesized TriFPP contained a
reactive fluorophosphonate to covalently modify reactive active
site serines, and biotin and rhodamine components for recovery
and recognition respectively (Figure 5B) and had been previ-
ously shown to label and selectively inactivate serine hydrolases
in Arabidopsis thaliana (11). Treatment of the semipurified
thioesterase with 10 µM TriFPP resulted in a 86% ( 3% (mean
( variation in replicates, n ) 3) inhibition of activity. When
the labeled protein was analyzed by SDS-PAGE, the 43 kDa
polypeptide could be seen to be labeled with the fluorescent
TriFPP confirming its identity as a hydrolase (Figure 5C). Based
on this, the protein was termed Passiflora edulis wall-bound
hydrolase (PeWH).
does support the identification of PeWH as such an enzyme.
First, as determined with PeWH, pectin acetyl esterases in a
range of Citrus species, including orange, lime, lemon, grapefruit
and clementine (18), are found in the exocarp, but not in the
flesh of the fruit. The purification of plant pectin acetyl esterases
have only been reported from orange and mung bean, with the
extraction techniques mirroring the procedures required to isolate
PeWH (19, 20). For example, high salt concentrations in
the extraction buffer (0.2-1 M NaCl) were required to solubilize
the ionically bound enzymes from the cell wall (19, 20). Like
PeWH, the pectin acetyl esterases are highly basic proteins and
are of a very similar molecular mass with the enzyme from
mung bean also being a 43 kDa protein (19, 21). The en-
dogenous functions of pectin acetyl esterases are not well
understood, though their activity in regulating the degree of
pectin acetylation, has been ascribed roles in softening fruit
tissue (18) and in controlling cell growth and elongation (21).
Interestingly, in Citrus fruit peel, pectin acetyl esterases were
localized at high concentrations in oil vesicles where functions
in wall modifications would seem improbable (18). Based on
our observations with PeWH, it is also possible that glycoside
hydrolases present in fruit peel are able to hydrolyze volatile
secondary metabolites. Such a role in natural product metabolism
may extend to many fruits. For example, in apples butyl esters
are hydrolyzed to release butanol into the surrounding air by
undefined esterases found at high concentrations in the cortex
and peel (22). In addition a recent study in the tropical snake
fruit (Salacca edulis) found that pectin methyl esterase activity
was linked to the metabolism of volatile methyl esters (23).
The diversity of roles adopted by glycoside hydrolases in fruit
ripening (24, 25) and their potential to act as bifunctional
enzymes contributing to the biogenesis of volatile secondary
metabolites is an interesting area for future study. Work will
now focus on cloning and expressing the enzyme both as a
means of confirming identity and for characterizing the enzyme
further.
The partially purified PeWH was then screened for activity
against the thioester substrates (Table 1). The greatest thioesterase
activity was determined with 2-methyl-3-furanthiol acetate (5),
with appreciable activities also determined with 8-acetylthio-
p-menthan-3-one (8). Reasonable thioesterase activity was also
determined with the putative passion fruit substrate 3-(acetylth-
io)hexyl acetate (4). In the case of compound 4, as determined
by GC analysis, PeWH was nine times more active in cleaving
the thioester bond than the carboxylester in this substrate.
Compounds 5-7, all contained the 5-membered furan ring, with
the PeWH thioesterase activity being much greater when the
acetyl-thioester was substituted at the 3 (5) rather than the 2
position (7). When the furan moiety became the acyl rather than
the alcohol moiety (6) all activity was lost. PeWH was also a
carboxylesterase, showing good activities toward p-nitrophe-
nylacetate (compound 10) and R-naphthyl acetate (9) but little
turnover of the umbelliferyl esters (11-13). As compared with
the commercially available hydrolases, PeWH showed some
interesting selectivity in its thioesterase activity. PeWH was
much more active toward the thioesters than the microbial lipase
and hydrolyzed the thioester 8-acetylthio-p-menthan-3-one (8)
with higher specific activity than porcine liver esterase. It was
however much less active as a carboxylesterase. In all the assays
it is likely that the true specific activity of PeWH was under-
estimated as the respective protein preparation was not totally
pure.
ABBREVIATIONS USED
PeWH, Passiflora edulis wall hydrolase; TriFPP, trifunctional
fluorophosphonate activity probe.
The 43 kDa putative thioesterase was then analyzed for its
physical properties. The PeWH polypeptide was shown to be
glycosylated, based on its reactivity to glycan staining (data not
shown). Similarly, when the semipurified protein from phenyl-
Superose chromatography was applied onto a concanavalin
A-Sepharose column, 100% of the activity was selectively
retained, with 91% recovered after elution with 0.1 M methyl-
R-D-mannopyranoside. When analyzed by isoelectric focusing
gel electophoresis, the PeWH migrated as a basic protein with
a pI between pH 9-10. The 43 kDa PeWH polypeptide was
excised from the SDS-PAGE gels of the final protein preparation
(Figure 4D) and digested with trypsin, prior to the analysis of
individual fractions by tandem MS based sequencing (12).
Sequence was obtained from five distinct peptides, which were
used to interrogate the plant genome and EST databases. From
the five major hits it was apparent the enzyme belonged to a
groupofproteinslabeledasfamily13glycosidehydrolases(16,17).
Of the proteins identified, pectin acetyl esterase (EC 3.1.1.6)
was the primary candidate, with three of the five fragments
generating significant hits matching this group of enzymes.
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Thioesterase Activity and Family 13 Glycoside Hydrolases.
Despite their potential importance in plant cell wall modifica-
tions, pectin acetyl esterases have not been well studied at the
biochemical level. However, what is known about these proteins