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.
Highlights
Chemical defence of marine organisms may be modulated through (i) pre-formed compounds that are constitutively present, (ii) de novo biosynthesis of defensive compounds that are formed following an attack by predators or microorganisms or (iii) biotransformation of defence molecules following disturbance of the cellular compartmentalization [1]
We report the partial purification and characterisation of nitrile hydratases isolated from the Mediterranean sponge A. cavernicola that convert aeroplysinin-1 (1) into the corresponding dienone amide (7) in vitro, it is not yet clear whether the enzymes are of sponge origin or produced by its symbiotic microorganisms
Enzyme activity was exclusively confined to the supernatants S1 and S2 indicating that the studied nitrile hydratase is a soluble rather than a membrane bound or membrane-associated enzyme (Figure 2)
Summary
Chemical defence of marine organisms may be modulated through (i) pre-formed compounds that are constitutively present, (ii) de novo biosynthesis of defensive compounds that are formed following an attack by predators or microorganisms or (iii) biotransformation of defence molecules following disturbance of the cellular compartmentalization [1]. A. aerophoba occurs in the Mediterranean Sea and in the Atlantic Ocean and like other species of this genus, accumulates brominated isoxazoline alkaloids such as the aerophobins, isofistularin-3 or aerothionin that may account for up to 10% of the dry weight [5,6] These compounds act as feeding deterrents against the marine fish Blennius sphinx and are thought to protect the sponges from predators [7]. In a second enzymatically catalyzed reaction that occurs in A. aerophoba and in A. cavernicola, the nitrile aeroplysinin-1 (1) is further converted to the corresponding dienone amide verongiaquinol (7) [4,8] Both aeroplysinin-1 (1) and the dienone amide show strong antibiotic activity against a broad spectrum of gram positive and gram negative marine and terrestrial bacteria and are believed to protect the sponge from infection with microbial pathogens at the site of wounding
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