Soybean Lipoxygenase-1 Oxidizes 3Z-Nonenal : A Route to 4S-Hydroperoxy-2E-Nonenal and Related Products

In previous work with soybean (Glycine max), it was reported that the initial product of 3Z-nonenal (NON) oxidation is 4-hydroperoxy-2E-nonenal (4-HPNE). 4-HPNE can be converted to 4-hydroxy-2E-nonenal by a hydroperoxide-dependent peroxygenase. In the present work we have attempted to purify the 4-HPNE-producing oxygenase from soybean seed. Chromatography on various supports had shown that O₂ uptake with NON substrate consistently coincided with lipoxygenase (LOX)-1 activity. Compared with oxidation of LOX's preferred substrate, linoleic acid, the activity with NON was about 400- to 1000-fold less. Rather than obtaining the expected 4-HPNE, 4-oxo-2E-nonenal was the principal product of NON oxidation, presumably arising from the enzyme-generated alkoxyl radical of 4-HPNE. In further work a precipitous drop in activity was noted upon dilution of LOX-1 concentration; however, activity could be enhanced by spiking the reaction with 13S-hydroperoxy-9Z,11E-octadecadienoic acid. Under these conditions the principal product of NON oxidation shifted to the expected 4-HPNE. 4-HPNE was demonstrated to be 83% of the 4S-hydroperoxy-stereoisomer. Therefore, LOX-1 is also a 3Z-alkenal oxygenase, and it exerts the same stereospecificity of oxidation as it does with polyunsaturated fatty acids. Two other LOX isozymes of soybean seed were also found to oxidize NON to 4-HPNE with an excess of 4S-hydroperoxy-stereoisomer.     

Discovery of a linoleate 9S-dioxygenase and an allene oxide synthase in a fusion protein of Fusarium oxysporum

Fusarium oxysporum is a devastating plant pathogen that oxidizes C₁₈ fatty acids sequentially to jasmonates. The genome codes for putative dioxygenase (DOX)-cytochrome P450 (CYP) fusion proteins homologous to linoleate diol synthases (LDSs) and the allene oxide synthase (AOS) of Aspergillus terreus, e.g., FOXB_01332. Recombinant FOXB_01332 oxidized 18:2n-6 to 9S-hydroperoxy-10(E),12(Z)-octadecadienoic acid by hydrogen abstraction and antarafacial insertion of molecular oxygen and sequentially to an allene oxide, 9S(10)-epoxy-10,12(Z)-octadecadienoic acid, as judged from nonenzymatic hydrolysis products (α- and γ-ketols). The enzyme was therefore designated 9S-DOX-AOS. The 9S-DOX activity oxidized C₁₈ and C₂₀ fatty acids of the n-6 and n-3 series to hydroperoxides at the n-9 and n-7 positions, and the n-9 hydroperoxides could be sequentially transformed to allene oxides with only a few exceptions. The AOS activity was stereospecific for 9- and 11-hydroperoxides with S configurations. FOXB_01332 has acidic and alcoholic residues, Glu⁹⁴⁶-Val-Leu-Ser⁹⁴⁹, at positions of crucial Asn and Gln residues (Asn-Xaa-Xaa-Gln) of the AOS and LDS. Site-directed mutagenesis studies revealed that FOXB_01332 and AOS of A. terreus differ in catalytically important residues suggesting that AOS of A. terreus and F. oxysporum belong to different subfamilies. FOXB_01332 is the first linoleate 9-DOX with homology to animal heme peroxidases and the first 9-DOX-AOS fusion protein.     

A Catalase-related Hemoprotein in Coral Is Specialized for Synthesis of Short-chain Aldehydes: DISCOVERY OF P450-TYPE HYDROPEROXIDE LYASE ACTIVITY IN A CATALASE*

In corals a catalase-lipoxygenase fusion protein transforms arachidonic acid to the allene oxide 8R,9-epoxy-5,9,11,14-eicosatetraenoic acid from which arise cyclopentenones such as the prostanoid-related clavulones. Recently we cloned two catalase-lipoxygenase fusion protein genes (a and b) from the coral Capnella imbricata, form a being an allene oxide synthase and form b giving uncharacterized polar products (Lõhelaid, H., Teder, T., Tõldsepp, K., Ekins, M., and Samel, N. (2014) PloS ONE 9, e89215). Here, using HPLC-UV, LC-MS, and NMR methods, we identify a novel activity of fusion protein b, establishing its role in cleaving the lipoxygenase product 8R-hydroperoxy-eicosatetraenoic acid into the short-chain aldehydes (5Z)-8-oxo-octenoic acid and (3Z,6Z)-dodecadienal; these primary products readily isomerize in an aqueous medium to the corresponding 6E- and 2E,6Z derivatives. This type of enzymatic cleavage, splitting the carbon chain within the conjugated diene of the hydroperoxide substrate, is known only in plant cytochrome P450 hydroperoxide lyases. In mechanistic studies using ¹⁸O-labeled substrate and incubations in H₂¹⁸O, we established synthesis of the C8-oxo acid and C12 aldehyde with the retention of the hydroperoxy oxygens, consistent with synthesis of a short-lived hemiacetal intermediate that breaks down spontaneously into the two aldehydes. Taken together with our initial studies indicating differing gene regulation of the allene oxide synthase and the newly identified catalase-related hydroperoxide lyase and given the role of aldehydes in plant defense, this work uncovers a potential pathway in coral stress signaling and a novel enzymatic activity in the animal kingdom.     

Appearance of new lipoxygenases in soybean cotyledons after germination and evidence for expression of a major new lipoxygenase gene

The appearance and subsequent disappearance of lipoxygenase activity at pH 6.8 in germinated cotyledons of soybean (Glycine max [L.]) was shown using a variant soybean cultivar (Kanto 101) that lacks the two lipoxygenase isozymes, L-2 and L-3, that are present in dry seeds of a normal soybean cultivar (Enrei). Three new lipoxygenases, designated lipoxygenase L-4, L-5, and L-6, were purified using anionic or cationic ion exchange chromatography. The major lipoxygenase in 5-day-old cotyledons of the variant soybean was lipoxygenase L-4. Lipoxygenases L-5 and L-6 preferentially produced 13(S)-hydroperoxy-9(Z), 11(E)-octadecadienoic acid (13S-HPOD) as a reaction product of linoleic acid, whereas lipoxygenase L-4 produced both 13S-HPOD and 9(S)-hydroperoxy-10(E), 12(Z)-octadecadienoic acid. All three isozymes have pH optima of 6.5, no activity at pH 9.0, and preferred linolenic acid to linoleic acid as a substrate. Partial amino acid sequencing of lipoxygenase L-4 showed that this isozyme shares amino acid sequence homology with lipoxygenases L-1, L-2, and L-3 but is not identical to any of them. This indicates that a new lipoxygenase, L-4, is expressed in cotyledons.     

Linoleate 9R-dioxygenase and allene oxide synthase activities of Aspergillus terreus

Oxygenation of linoleic acid by Aspergillus terreus was studied with LC-MS/MS. 9(R)-Hydroperoxy-10(E),12(Z)-octadecadienoic acid (9R-HpODE) was identified along with 10(R)-hydroxy-8(E),12(Z)-octadecadienoic acid and variable amounts of 8(R)-hydroxy-9(Z),12(Z)-octadecadienoic acid. 9R-HpODE was formed from [11S-²H]18:2n − 6 with loss of the deuterium label, suggesting antarafacial hydrogen abstraction and oxygenation. Two polar metabolites were identified as 9-hydroxy-10-oxo-12(Z)-octadecenoic acid (α-ketol) and 13-hydroxy-10-oxo-11(E)-octadecenoic acid (γ-ketol), likely formed by spontaneous hydrolysis of an unstable allene oxide, 9(R),10-epoxy-10,12(Z)-octadecadienoic acid. α-Linolenic acid and 20:2n − 6 were oxidized to hydroperoxy fatty acids at C-9 and C-11, respectively, but α- and γ-ketols of these fatty acids could not be detected. The genome of A. terreus lacks lipoxygenases, but contains genes homologous to 5,8-linoleate diol synthases and linoleate 10R-dioxygenases of aspergilli. Our results demonstrate that linoleate 9R-dioxygenase linked to allene oxide synthase activities can be expressed in fungi.     

Cationic substrates of soybean lipoxygenase-1

Soybean lipoxygenase-1 (SBLO-1) catalyzes the oxygenation of 1,4-dienes to produce conjugated diene hydroperoxides. The best substrates are anions of fatty acids; for example, linoleate is converted to 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoate. The manner in which SBLO-1 binds substrates is uncertain. In the present work, it was found that SBLO-1 will oxygenate linoleyltrimethylammonium ion (LTMA) to give primarily13(S)-hydroperoxy-9(Z),11(E)-octadecadienyltrimethylammonium ion. The rate of this process is about the same at pH 7 and pH 9 and is about 30% of the rate observed with linoleate at pH 9. At pH 7, SBLO-1 oxygenates linoleyldimethylamine (LDMA) to give primarily 13(S)-hydroperoxy-9(Z),11(E)-octadecadienyldimethylamine. The oxygenation of LDMA occurs at about the same rate as LTMA at pH 7, but more slowly at pH 9. The results demonstrate that SBLO-1 will readily oxygenate substrates in which the carboxylate of linoleate is replaced with a cationic group, and the products of these reactions have the same stereo- and regiochemistry as the products obtained from fatty acid substrates.     

Vanadium-catalyzed transformation of 13(S)-hydroperoxy-9(Z), 11(E)-octadecadienoic acid: Structural studies on epoxy alcohols and trihydroxy acids

Treatment of methyl 13(S)-hydroperoxy-9(Z), 11(E)-octadecadienoate with vanadium oxyacetylacetonate led to the formation of two diastereometric α,β-epoxy alcohols, i.e. methyl 11(R), 12(R)-epoxy-13(S)-hydroxy-9(Z)-octadecenoate and methyl 11(S), 12(S)-epoxy-13(S)-hydroxy-9(Z)-octadecenoate. The epoxy alcohols underwent spontaneous hydrolysis into isomeric trihydroxyesters. The first mentioned epoxy alcohol afforded methyl 9(R), 12(S), 13(S)- and methyl 9(S), 12(S), 13(S)-trihydroxy-10(E)-octadecenoates as major hydrolysis products whereas the latter epoxy alcohol afforded methyl 9(R), 12(R), 13(S)- and methyl 9(S), 12(R)-13(S)-trihydroxy-10(E)-octadecenoates as major compounds. Smaller amounts of diastereomeric methyl 11,12,13-trihydroxy-9-octadecenoates were also formed from both epoxy alcohols. The vanadium-catalyzed conversion of 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoic acid (13(S)HPOD) (methyl ester) into α,β-epoxy alcohols and their further conversion into trihydroxy derivatives offers a model system for similar transformations of certain poly-unsaturated fatty acids recently described in the fungus, Saprolegnia parasitica.     

Isolation and Characterization of Two Geometric Allene Oxide Isomers Synthesized from 9S-Hydroperoxylinoleic Acid by Cytochrome P450 CYP74C3: STEREOCHEMICAL ASSIGNMENT OF NATURAL FATTY ACID ALLENE OXIDES*

Specialized cytochromes P450 or catalase-related hemoproteins transform fatty acid hydroperoxides to allene oxides, highly reactive epoxides leading to cyclopentenones and other products. The stereochemistry of the natural allene oxides is incompletely defined, as are the structural features required for their cyclization. We investigated the transformation of 9S-hydroperoxylinoleic acid with the allene oxide synthase CYP74C3, a reported reaction that unexpectedly produces an allene oxide-derived cyclopentenone. Using biphasic reaction conditions at 0 °C, we isolated the initial products and separated two allene oxide isomers by HPLC at −15 °C. One matched previously described allene oxides in its UV spectrum (λ_max 236 nm) and NMR spectrum (defining a 9,10-epoxy-octadec-10,12Z-dienoate). The second was a novel stereoisomer (UV λ_max 239 nm) with distinctive NMR chemical shifts. Comparison of NOE interactions of the epoxy proton at C9 in the two allene oxides (and the equivalent NOE experiment in 12,13-epoxy allene oxides) allowed assignment at the isomeric C10 epoxy-ene carbon as Z in the new isomer and the E configuration in all previously characterized allene oxides. The novel 10Z isomer spontaneously formed a cis-cyclopentenone at room temperature in hexane. These results explain the origin of the cyclopentenone, provide insights into the mechanisms of allene oxide cyclization, and define the double bond geometry in naturally occurring allene oxides.     

Acid-catalyzed transformation of 13(S)-hydroperoxylinoleic acid into epoxyhydroxyoctadecenoic and trihydroxyoctadecenoic acids

Acid treatment of (13S)-(9Z,11E)-13-hydroperoxy-9,11-octadecadienoic acid in tetrahydrofuran-water solvent afforded mainly (11R,12R,13S)-(Z)-12,13-epoxy-11-hydroxy-9-octadecenoic acid, diastereomeric (Z)-11,12,13-trihydroxy-9-octadecenoic acids and four isomers of (E)-9,12,13(9,10,13)-trihydroxy-10(11)-octadecenoic acid. Other minor products were oxooctadecadienoic, (E)-9(13)-hydroxy-13(9)-oxo-10(11)-octadecenoic and (E)-12-oxo-10-dodecenoic acids. A heterolytic mechanism for acid catalysis was indicated, even though most of the products characterized also have been observed as a result of homolytic decomposition of the hydroperoxide via an oxy radical. Most of the products found in this study have been observed as metabolites of (13S)-(9Z,11E)-13-hydroperoxy-9,11-octadecadenoic acid in biological systems, and analogous compounds have been reported as metabolites of (12S)-(5Z,8Z,10E, 14Z)-12-hydroperoxy-5,8,10,14-hydroperoxy-5,8,10,14-eicosatetraenoic acid in either blood platelets or lung tissue.     

Geometrical Configuration of 12,13-Epoxyoctadeca-9,11-dienoic Acid,a Product of the Reaction Catalyzed by Flaxseed Allene Oxide Synthase (CYP74A)

The geometrical configuration of a short-living allene oxide reaction product that arises under the catalysis by flaxseed allene oxide synthase (CYP74A) was studied by NMR spectroscopy. The structure of (9Z, 11E)-12,13-epoxyoctadeca-9,11-dienoic acid was established for it from the results of the nuclear Overhauser effect measurements.     

Separation of enzymatic functions and variation of spin state of rice allene oxide synthase-1 by mutation of Phe-92 and Pro-430

Rice allene oxide synthase-1 mutants carrying F92L, P430A or F92L/P430A amino acid substitution mutations were constructed, recombinant mutant and wild type proteins were purified and their substrate preference, UV–vis spectra and heme iron spin state were characterized. The results show that the hydroperoxide lyase activities of F92L and F92L/P430A mutants prefer 13-hydroperoxy substrate to other hydroperoxydienoic acids or hydroperoxytrienoic acids. The Soret maximum was completely red-shifted in P430A and F92L/P430A mutants, but it was partially shifted in the F92L mutant. ESR spectral data showed that wild type, F92L and P430A mutants occupied high and low spin states, while the F92L/P430A mutant occupied only low spin state. The extent of the red shift of the Soret maximum increased as the population of low spin heme iron increased, suggesting that the spectral shift reflects the high to low transition of heme iron spin state in rice allene oxide synthase-1. Relative to wild type allene oxide synthase-1, the hydroperoxide lyase activities of F92L and F92L/P430A are less sensitive to inhibition by imidazole with (13S or 9S)-hydroperoxydienoic acid as substrate and more sensitive than wild type with (13S)-hydroperoxytrienoic acid as substrate. Our results suggest that hydroperoxydienoic acid is the preferred substrate for the hydroperoxide lyase activity and (13S)-hydroperoxytrienoic acid is the preferred substrate for allene oxide synthase activity of allene oxide synthase-1.     

Double function hydroperoxide lyases/epoxyalcohol synthases (CYP74C) of higher plants: identification and conversion into allene oxide synthases by site-directed mutagenesis

The CYP74C subfamily of fatty acid hydroperoxide transforming enzymes includes hydroperoxide lyases (HPLs) and allene oxide synthases (AOSs). This work reports a new facet of the putative CYP74C HPLs. Initially, we found that the recombinant CYP74C13_MT (Medicago truncatula) behaved predominantly as the epoxyalcohol synthase (EAS) towards the 9(S)-hydroperoxide of linoleic acid. At the same time, the CYP74C13_MT mostly possessed the HPL activity towards the 13(S)-hydroperoxides of linoleic and α-linolenic acids. To verify whether this dualistic behaviour of CYP74C13_MT is occasional or typical, we also examined five similar putative HPLs (CYP74C). These were CYP74C4_ST (Solanum tuberosum), CYP74C2 (Cucumis melo), CYP74C1_CS and CYP74C31 (both of Cucumis sativus), and CYP74C13_GM (Glycine max). All tested enzymes behaved predominantly as EAS toward 9-hydroperoxide of linoleic acid. Oxiranyl carbinols such as (9S,10S,11S,12Z)-9,10-epoxy-11-hydroxy-12-octadecenoic acids were the major EAS products. Besides, the CYP74C31 possessed an additional minor 9-AOS activity. The mutant forms of CYP74C13_MT, CYP74C1_CS, and CYP74C31 with substitutions at the catalytically essential domains, namely the “hydroperoxide-binding domain” (I-helix), or the SRS-1 domain near the N-terminus, showed strong AOS activity. These HPLs to AOSs conversions were observed for the first time. Until now a large part of CYP74C enzymes has been considered as 9/13-HPLs. Notwithstanding, these results show that all studied putative CYP74C HPLs are in fact the versatile HPL/EASs that can be effortlessly mutated into specific AOSs.     

One-pot synthesis of bioactive cyclopentenones from α-linolenic acid and docosahexaenoic acid

Oxidation products of the poly-unsaturated fatty acids (PUFAs) arachidonic acid, α-linolenic acid and docosahexaenoic acid are bioactive in plants and animals as shown for the cyclopentenones prostaglandin 15d-PGJ₂ and PGA₂, cis-(+)-12-oxophytodienoic acid (12-OPDA), and 14-A-4 neuroprostane. In this study an inexpensive and simple enzymatic multi-step one-pot synthesis is presented for 12-OPDA, which is derived from α-linolenic acid, and the analogous docosahexaenoic acid (DHA)-derived cyclopentenone [(4Z,7Z,10Z)-12-[[-(1S,5S)-4-oxo-5-(2Z)-pent-2-en-1yl]-cyclopent-2-en-1yl] dodeca-4,7,10-trienoic acid, OCPD]. The three enzymes utilized in this multi-step cascade were crude soybean lipoxygenase or a recombinant lipoxygenase, allene oxide synthase and allene oxide cyclase from Arabidopsis thaliana. The DHA-derived 12-OPDA analog OCPD is predicted to have medicinal potential and signaling properties in planta. With OCPD in hand, it is shown that this compound interacts with chloroplast cyclophilin 20-3 and can be metabolized by 12-oxophytodienoic acid reductase (OPR3) which is an enzyme relevant for substrate bioactivity modulation in planta.     

Double hydroperoxidation of α-linolenic acid by potato tuber lipoxygenase

The potato tuber lipoxygenase preparations convert α-linolenic acid not only to 9(S)-HPOTE, but also to some more polar metabolites. Two of these polar products, I and II, with ultraviolet absorbance maxima at 267 nm were purified by HPLC. It was found that metabolites I and II have, respectively, one and two hydroperoxy groups. Products of NaBH₄ reduction of both I and II were identified by their chemical ionization and electron impact mass spectra and by ¹H-NMR spectra as 9,16-dihydroxy-10(E), 12(Z), 14(E)-octadecatrienoic acid. The obtained results suggest that compound II is 9,16-dihydroperoxy-10(E), 12(Z), 14(E)-octadecatrienoic acid and product I is a mixture of two positional isomers, 9-hydroxy-16-hydroperoxy-10(E),12(Z),14(E)-octadecatrienoic and 9-hydroperoxy-16-hydroxy-10(E),12(Z), 14(E)-octadecatrienoic acids. Lipoxygenase converts efficiently [¹⁴C]9-HOTE into product I. Also, both metabolites I and II are the products of double dioxygenation. The second oxygenation at C-16 position as well as the first one at C-9 is controlled by lipoxygenase.     

Detection of unprecedented allene oxide synthase member of CYP74B subfamily: CYP74B33 of carrot (Daucus carota)

Enzymes of CYP74 family widespread in higher plants control the metabolism of fatty acid hydroperoxides to numerous bioactive oxylipins. Hydroperoxide lyases (HPLs, synonym: hemiacetal synthases) of CYP74B subfamily belong to the most common CYP74 enzymes. HPLs isomerize the hydroperoxides to the short-lived hemiacetals, which are spontaneously decomposed to aldehydes and aldoacids. All CYP74Bs studied yet except the CYP74B16 (flax divinyl ether synthase, LuDES) possessed the 13-HPL activity. Present work reports the cloning of the expressed CYP74B33 gene of carrot (Daucus carota L.) and studies of catalytic properties of the recombinant CYP74B33 protein. In contrast to all CYP74B proteins studied yet, CYP74B33 behaved differently in few respects. Firstly, the preferred substrates of CYP74B33 are 9-hydroperoxides. Secondly and most importantly, CYP74B33 exhibits the 9-allene oxide synthase (AOS) activity. For example, the 9(S)-hydroperoxide of linoleic acid (9-HPOD) underwent the conversion to α-ketol via the short-lived allene oxide. Uncommonly, the 9-HPOD conversion affords a minority of cis-10-oxo-11-phytoenoic acid, which is also produced by CYP74C but not the CYP74A AOSs. The similar product patterns were observed upon the incubations of CYP74B33 with 9(S)-hydroperoxide of α-linolenic acid. The enzyme possessed a mixed HPL, AOS, and the epoxyalcohol synthase activity toward the 13-hydroperoxides, but the total activity was much lower than toward 9-hydroperoxides. Thus, the obtained results show that CYP74B33 is an unprecedented 9-AOS within the CYP74B subfamily.     

Engineered heterologous FPP synthases-mediated Z,E-FPP synthesis in E. coli

Production of Z-type farnesyl diphosphate (FPP) has not been reported in Escherichia coli. Here we present the fusion enzyme (ILRv) of E. coli E,E-FPP synthase (IspA) and Mycobacterium tuberculosis Z,E-FPP synthase (Rv1086), which can produce primarily Z,E-FPP rather than E,E-FPP, the predominant stereoisomer found in most organisms. Z,E-farnesol (FOH) was produced from E. coli harboring the bottom portion of the MVA pathway and the fusion FPP synthase (ILRv) at a titer of 115.6 mg/L in 2 YT medium containing 1% (v/v) glycerol as a carbon source and 5 mM mevalonate. The Z,E-FOH production was improved by 15-fold, compared with 7.7 mg/L obtained from the co-overexpression of separate IspA and Rv1086. The Z,E-FPP was not metabolized in native metabolic pathways of E. coli. It would be of interest to produce Z,E-FPP derived sesquiterpenes from recombinant E. coli due to no loss of Z,E-FPP substrate in endogenous metabolism of the host strain.     

Codon Optimisation Is Key for Pernisine Expression in Escherichia coli

Background

Pernisine is an extracellular serine protease from the hyperthermophilic Archaeon Aeropyrum pernix K1. Low yields from the natural host and expression problems in heterologous hosts have limited the potential applications of pernisine in industry.

Methodology/ Principal Findings

The challenges of pernisine overexpression in Escherichia coli were overcome by codon preference optimisation and de-novo DNA synthesis. The following forms of the pernisine gene were cloned into the pMCSGx series of vectors and expressed in E. coli cells: wild-type (pernisine^wt), codon-optimised (pernisine^co), and codon-optimised with a S355A mutation of a predicted active site (pernisine^S355Aco). The fusion-tagged pernisines were purified using fast protein liquid chromatography equipped with Ni²⁺ chelate and gel filtration chromatography columns. The identities of the resultant proteins were confirmed with N-terminal sequencing, tandem mass spectrometry analysis, and immunodetection. Pernisine^wt was not expressed in E. coli at detectable levels, while pernisine^co and pernisine^S355Aco were expressed and purified as 55-kDa proforms with yields of around 10 mg per litre E. coli culture. After heat activation of purified pernisine, the proteolytic activity of the mature pernisine^co was confirmed using zymography, at a molecular weight of 36 kDa, while the mutant pernisine^S355Aco remained inactive. Enzymatic performances of pernisine evaluated under different temperatures and pHs demonstrate that the optimal enzymatic activity of the recombinant pernisine is ca. 100°C and pH 7.0, respectively.

Conclusions/ Significance

These data demonstrate that codon optimisation is crucial for pernisine overexpression in E. coli, and that the proposed catalytic Ser355 has an important role in pernisine activity, but not in its activation process. Pernisine is activated by autoproteolytical cleavage of its N-terminal proregion. We have also confirmed that the recombinant pernisine retains the characteristics of native pernisine, as a calcium modulated thermostable serine protease.