Karenia brevis
Creators: name: Sun, Pengfei
name: Leeson, Cristian
name: Zhi, Xiaoduo
name: Leng, Fenfei
name: Pierce, Richard H.
name: Henry, Michael S.
name: Rein, Kathleen S.
Publisher: name: Zenodo
Year: 2016
Subjects: Biodiversity
Taxonomy
Protista
Myzozoa
Insecta
Hemiptera
Cicadidae
Karenia
Karenia brevis

description: 2.3. Characterization of K. brevis EH
Fluorescent probe sets 3b / 5b and 3a / 5a were used for initial characterization of the expressed K. brevis EH (Fig. 3C). The EH was optimally active at pH 6.8–7.1, with half-maximal activity at pH 6.5 and 8.3. Consistent with the selectivity observed in the K. brevis CFE, the purified EH showed a preference for epoxide 5b over 5a. The ratio of initial rates (V o) for substrates 5a: 5b was 1:4. Epoxide hydrolases of the a,b- hydrolase type share significant homologies with other hydrolases, including esterases. As done with the CFE, EH activity was distinguished from esterase activity using the substrates 3a and 5a. No activity was observed using the esterase probes indicating that the expressed protein was indeed an EH.
Substrate and enantioselectivity of the K. brevis EH was assessed by kinetic studies. Kinetic parameters of the enzyme using substrates 5a and 5b were performed using the sensitive fluorescent assay. Other substrates having no fluorescent reporter group were evaluated by GC–MS analysis of the reaction mixtures using 1- naphthol as an internal standard. The data shown in Table 1 indicates that the K. brevis EH has a strong preference for terminal epoxides over internal epoxides (Entries 3–9 vs. Entries 1–2). Aliphatic substituents are preferred over aromatic substituents by approximately 5:1 (Entries 7–9 vs. Entries 3–6) and the enzyme shows only a slight preference for R over S enantiomers (Entries 3 and 4). The reaction rates using 1,2-disubstituted epoxides as substrates are too slow to be determined using the GC–MS method. However, disubstituted epoxides proved to be inhibitors of the K. brevis EH only when an aromatic substituent is present (Entries 10–12 vs. Entries 13–14) and cis -disubstituted epoxides are more potent inhibitors than those that are trans -disubstituted (Entries 10–12).
A noteworthy distinction between the monensin (2) pathway and the proposed brevetoxin (1) pathway is the site of nucleophilic attack on the epoxide substrate (Scheme 1). In the case of non-ladder polyethers, the majority of epoxide ring opening reactions proceed via attack at the proximal carbon (exo-tet cyclization) while polyether ladder formation requires attack at the distal carbon (endo-tet cyclization). Both the empirical Baldwin’s rules (Baldwin, 1976) and molecular orbital calculations (Gruber et al., 1999) indicate that the endo-tet cyclization pathway is kinetically disfavored. The exception among the bacterial polyketides is lasalocid. In this polyether, the pyran ring arises from the endo-tet cyclization of an intermediate epoxide. This reaction is catalyzed by the EH Lsd19. Lsd19 is a bifunctional enzyme consisting of two domains (A and B). Each domain is responsible for the cyclization of one of the rings with Lsd19B catalyzing the endo-tet cyclization of the terminal ring (Minami et al., 2011).
The ability of the K. brevis EH to perform cyclization was also evaluated. The substrate trans -4,5-epoxy-hexanol 9 was prepared as previously described (Coxon et al., 1973).This substrate may undergo endo-tet cyclization to produce pyran 10, or exo-tet cyclization to produce furan 11 (Scheme 4).The substrate was incubated with the expressed EH in reaction buffer and the reaction was monitored by GC–MS. Both possible products were identified by GC–MS, by comparison with authentic samples however, neither the percent conversion nor the product ratio differed from that of the buffer alone.
description: Published as part of Sun, Pengfei, Leeson, Cristian, Zhi, Xiaoduo, Leng, Fenfei, Pierce, Richard H., Henry, Michael S. & Rein, Kathleen S., 2016, Characterization of an epoxide hydrolase from the Florida red tide dinoflagellate, Karenia brevis, pp. 11-21 in Phytochemistry 122 on pages 15-16, DOI: 10.1016/j.phytochem.2015.11.002, http://zenodo.org/record/10485359