Characterization of the substrate specificity of squalene-hopene cyclases (SHCs) (2013)

Cyclic terpenoids form a large group of natural products with various biological functions. About 60,000 different cyclic terpenoids have been identified by now, containing scaffolds of ten to more than 30 carbon atoms. Among this huge amount of different cyclic compounds, there are many well-known flavors and fragrances, such as menthol or limonene, compounds which are widely used for pharmaceutical purposes like the antitumor compound Taxol and the anti-malaria agent artemisinin, or common membrane constituents and hormones such as the sterols. All of these natural products are derived from cyclization reactions of few linear precursor molecules catalyzed by terpenoid cyclases. The main focus of the present work rests on one very interesting family of the terpenoid cyclases, the squalene-hopene cyclases (SHCs). Among the over 300 annotated SHCs, most extensive studies had been carried out characterizing the SHC from the thermophilic bacterium Alicyclobacillus acidocaldarius (AacSHC), solving the crystal structure and the complex cyclization mechanism of the C30 precursor squalene into the pentacyclic products hopene and hopanol. This reaction constitutes one of the most complex reaction mechanisms found in nature, including the stereospecific formation of nine stereocenters and 13 covalent bonds.13 Besides AacSHC, several SHCs were partially characterized in previous works. Our interest was triggered by the ethanol and sugar tolerant strain Zymomonas mobilis which is known as one of the most potent hopanoid producers. Zymomonas mobilis contains two genes encoding for SHCs: ZmoSHC1 and the formerly partially characterized ZmoSHC2. It could be shown that the SHCs are also capable of cyclizing other linear terpenoids. For example, it was found that truncated squalene analogs were accepted as substrates by AacSHC and also the alcohol homofarnesol could be converted into the corresponding cyclic ether ambroxan.16–19 These results indicate that SHCs represent a promising family for catalysis of very different and complex cyclization reactions. Thus, we decided to investigate the SHCs’ potentials regarding their substrate specificities. In order to characterize the squalene-hopene cyclases ZmoSHC1 and ZmoSHC2 and compare them with AacSHC especially regarding their biocatalytic activities towards unnatural substrates, the SHCs were cloned and heterologously expressed in Escherichia coli. Functional expression was confirmed by conversion of the natural substrate squalene. For the direct comparison, a protocol for partial purification of the membrane-anchored SHCs was elaborated and optimized. For this partial purification as well as for the conversion of the hydrophobic substrates in aqueous milieu a suitable detergent had to be selected. ZmoSHC1 was characterized in more detail, retrieving information about pH- and temperature-dependence of the activity and the biocatalytic stability over a long period of time as well as inhibitory effects. All of the three enzymes were tested with unnatural substrates of C10-C18 carbon chain lengths. A special focus was laid on substrates containing functional groups such as hydroxyl , carboxy- or keto-groups expected to participate in the cyclization reaction, as shown for the hydroxyl-group of homofarnesol. Several of the substrates were accepted and cyclic products were generated. Interestingly, the functional groups were integrated in the final ring closure and to products with new properties were obtained. Homofarnesol conversion yielding the cyclic ether ambroxan, which is known as a expensive and rare flavor compound, was observed as reported in the literature. Also the corresponding carboxylic acid, homofarnesoic acid, could be converted into the cyclic lactone sclareolide. The C15 tertiary alcohol nerolidol was accepted as substrate and the bicyclic ether caparrapioxide was formed. Lastly, two ketones were accepted as substrates leading to cyclic enol ether products. Within the present work, all of these new products were characterized after preparative biotransformation and product isolation. Not only the facts that these substrates are much shorter than the natural substrate squalene and possess different functional groups which take part in the cyclization reaction and that useful products containing new properties are formed, but also the different activities of the SHCs towards these substrates are remarkable. Thus, it could be shown that ZmoSHC1 exhibits special biocatalytic properties, as the substrate activity pattern was unexpected. While squalene was converted very poorly, good activity was found towards the reaction of homofarnesol to ambroxan. All of the other substrates were converted in low but significant rates into the corresponding cyclic products. A completely different substrate activity pattern was observed using AacSHC as biocatalyst. Besides very good squalene conversion, much lower activities towards all of the other substrates were found. Using ZmoSHC2, only very low conversion rates were found for squalene and farnesylacetone and no conversion of any of the other substrates. Based on these observations, it can be concluded that ZmoSHC1 represents a versatile biocatalyst for complex cyclization reactions, as it shows unexpected substrate activity towards other substrates than squalene. In the present work, these and further detailed results are described. Besides the examination of the SHCs’ activities towards different substrates there were also several mutants created in order to find explanations for the differences between the SHCs regarding their substrate activities. This characterization of the triterpenoid cyclase ZmoSHC1 and discussion of their special properties leads to new conclusions about the potential of SHCs to serve as potent biocatalysts for new reactions.