An aeroplysinin-1 specific nitrile hydratase isolated from the marine sponge Aplysina cavernicola

Abstract

A nitrile hydratase (NHase) that specifically accepts the nitrile aeroplysinin-1 (1) as a substrate and converts it into the dienone amide verongiaquinol (7) was isolated, partially purified and characterized from the Mediterranean sponge Aplysina cavernicola; although it is currently not known whether the enzyme is of sponge origin or produced by its symbiotic microorganisms. The formation of aeroplysinin-1 and of the corresponding dienone amide is part of the chemical defence system of A. cavernicola. The latter two compounds that show strong antibiotic activity originate from brominated isoxazoline alkaloids that are thought to protect the sponges from invasion of bacterial pathogens. The sponge was shown to contain at least two NHases as two excised protein bands from a non denaturating Blue Native gel showed nitrile hydratase activity, which was not observed for control samples. The enzymes were shown to be manganese dependent, although cobalt and nickel ions were also able to recover the activity of the nitrile hydratases. The temperature and pH optimum of the studied enzymes were found at 41 °C and pH 7.8. The enzymes showed high substrate specificity towards the physiological substrate aeroplysinin-1 (1) since none of the substrate analogues that were prepared either by partial or by total synthesis were converted in an in vitro assay. Moreover de-novo sequencing by mass spectrometry was employed to obtain information about the primary structure of the studied NHases, which did not reveal any homology to known NHases.

Figure 1

Biotransformation of the isoxazoline alkaloids as described by Teeyapant and Proksch [4] with isofistularin-3 as an example.

Figure 2

Decrease of the substrate aeroplysinin-1 (in %) relative to a control without enzyme for fractions derived from differential centrifugation at 800 g and 100,000 g. The supernatant S1 and pellet P1 resulted from a centrifugation step at 800 g, while the supernatant S2 and pellet P2 were obtained from a centrifugation step at 100,000 g. Activity (in %) is defined as the decrease of substrate relative to a control lacking the enzyme fraction. The activity is calculated for 10 µg of protein. (One representative experiment shown).

Figure 3

Decrease of the substrate aeroplysinin-1 (in %) relative to a control without enzyme for the supernatant S2 (positive control), a heat inactivated sample, a dialysed sample, a dialysed sample plus a heat inactivated sample and a dialysed sample with 7.5 mM Mn²⁺. Activity (in %) is defined as the decrease of substrate relative to a control without enzyme. The activity is calculated for 10 µg of protein. The experiments were performed in triplicate (n = 3) and the error bars are defined as standard deviation.

Figure 4

Blue Native-PAGE (A) and SDS-PAGE (B) analysis of typical size exclusion chromatography fractions. In (A), a Coomassie stained NativePAGE 4%–16% Bis-Tris gel of typical SEC fractions is shown. The molecular weights of reference proteins are depicted on the left. (B) shows a Coomassie stained 12% SDS-PAGE of the same fractions as in (A). The molecular weights of the standard proteins are given next to the gel. Above the gels in (A) and (B), the specific activity (in U/mg) of the respective fraction with the HPLC based assay (see methods) are shown.

Figure 5

Decrease of the substrate aeroplysinin-1 (in %) relative to a control without enzyme for gel segments excised from the protein bands at 146 and 242 kDa (SEC fraction equivalent to fraction 14 in Figure 4; n = 1) in Blue Native-PAGE as well as other parts of the gel (control; n = 3). The activity is defined as the decrease of substrate relative to a measurement without gel. The error bars are defined as standard deviation.

Figure 6

(A) Temperature-dependence of the activity of the purified enzyme (SEC fraction 14 or equivalent; n = 3). The data were analysed assuming a Gaussian distribution. The activity (in %) is defined as the decrease of substrate relative to a control without protein. The activity is calculated for 0.1 µg of protein. The error bars are defined as standard deviation. (B) Influence of the pH on the activity of the nitrile hydratases in the supernatant S2. The activity (in %) is defined as the decrease of substrate relative to a control without protein. The activity is calculated for 10 µg of protein.

Figure 7

(A) Influence of selected metal ions (each at a concentration of 7.5 mM) on the activity of the purified nitrile hydratases (SEC fraction 14 or equivalent; n = 3). (B) Influence of different concentrations of Mn²⁺ on the activity of the purified enzyme (SEC fraction 14 or equivalent; n = 3). The data were analysed assuming a one site binding model, which resulted in an apparent affinity constant of 0.856 ± 0.228 mM for Mn²⁺ ions. The activity is defined as the decrease of substrate (in %) relative to a control without protein. The activity is calculated per 0.1 µg of protein. The error bars are defined as standard deviation.

Figure 8

Aeroplysinin-1 and its derivatives.

Figure 9

(A) The turnover of aeroplysinin-1 and selected derivatives by the purified enzyme (SEC fraction 14 or equivalent; n = 3); (B) Competition between aeroplysinin-1 and its derivatives (at equimolar concentrations with SEC fraction 14 or equivalent; n = 3). The activity (in %) is defined as the decrease of substrate relative to a control without enzyme. The activity is calculated per 0.1 µg of protein. The error bars are defined as standard deviation.