Alicyclobacillus

Currently there is no experimentally determined three-dimensional structural information available for OSCs, although studies with a related enzyme, squa-lene-hopene cyclase (SC EC 5.4.99.7) have proved informative. SCs are involved in the direct cyclisation of squalene to pentacyclic triterpenoids known as hopanoids, which play an integral role in membrane structure in prokaryotes [ 51 ]. A number of SC genes have been cloned from bacteria [52 - 54]. The SC and OSC enzymes have related predicted amino acid sequences, and so should have similar spatial structures [55]. The crystal structure of recombinant SC from the Gram-positive bacterium Alicyclobacillus acidocaldarius has established that the enzyme is dimeric [55]. Each subunit consists of two a-a barrel domains that assemble to form a central hydrophobic cavity [55,56]. 

Alicyclobacillus acidocaldarius SC amino acid sequence (amino acids 376 and 377 of the DDTAV motif) are also essential for enzyme activity [59]. These residues are located in the large central cavity of the dimeric enzyme [55]. Interestingly, targeted mutations that convert the DDTAV motif of SC to DCTAE (the corresponding OSC motif) result in a change in substrate specificity from squalene to 2,3-oxidosqualene [61]. 

Alicyclobacillus Squalene-hopene cyclase (SC). . raveyllscEkdeByI.. mekirryllheBredBt. . ypdvBdBavvvwa.. rraveylkreBkpdBsB.. kaldwveqhBnpdBgB.. acidocaldarius... 

Walls, I. and Chuyate, R. (2000a) Spoilage of fruit juices by Alicyclobacillus acidoterrestris. Food Australia 52(7), 286-88. 

In this context, Hoshino et al. have recently reported the cyclization of (3S)-2,3-oxidosqualene catalyzed by a Gly600-deletion mutant (AG600SHC) of the enzyme squalene-hopene cyclase (SHC) from Alicyclobacillus acidocal-darius (Scheme 31) [108]. The enzymatic biotransformation gave monocyclic (9) and tricyclic triterpenoids (38 and 44-47), but no detectable bicychc products [108]. Despite the authors biogenetic proposal of a conventional carbocationic pathway, the skeletal profile of products reported closely resembles that expected for a radical-type cyclization. 

Comparable studies have been performed for the formation of co-cyclohexyl fatty acids in Alicyclobacillus acidocaldarius and the pathways are identical [100]. A recent publication concerning this later pathway has shown that the final remaining stereochemical ambiguity, the stereochemistry of proton loss at C-6 in the initial 1,4-conjugate elimination of shikimate occurs with loss of the pro-6R proton [102]. This mirrors the stereochemistry of normal shikimate metabolism in the formation of chorismate from 5-enolpyruvyl-shikimate 3-phosphate. 

In 1997, Georg E. Schulz in Freiburg succeeded in determining the structure of the membrane-bound squalene-hopene cyclase from the thermophilic microorganism Alicyclobacillus acidocaldar ms (Fig. 3.53 and Fig. 3.54). [258] The enzyme consists of two domains of approximately equal size. Between the two is a cavity with a volume of 1200 A, which contains the active site this is accessible through a non-polar channel in domain 2 (Fig. 3.55). 

Example 1 Screening of Fruit Juices Spoilage by Alicyclobacillus spp. 

Contamination by Alicyclobacillus spp. was firstly detected in apple juice but, since then, a larger variety of fruit juices, soft drink, fortified with minerals products have been found to be contaminated. A. acidoterrestris and A. acidocaldarius are the most common specie able to cause typical off-fiavors (medicine-like taints) in fruit juices this was related to the production of 2-methoxyphenol (guaiacol), 2-6-dibromophenol, 2-6 diclorophenol [62, 63] which are retained to be the markers of contamination. 

Different types of commercial fruit juices (orange, peach, pear and apple) artificially contaminated by Alicyclobacillus spp. were tested with the EOS in an experimental work that last for almost two years. 

Preliminary results reported in Gobbi et ai. [55] were very promising, showing that EOS has good detection capabilities, being able to early reveal the presence of Alicyclobacillus spp. just after a growth time of 24 h. The sensors showed some specificity related with the juice matrix, indeed contamination was easily identified in orange and peach juices (where detection threshold was around lOOcfu/ml) whereas it was impossible to correctly classify the contaminated apple juice samples. 

M. Walker, C.A. PhilUps, Alicyclobacillus acidoterrestris an increasing threat to the fruit juice industry Int J. Food Sci. Technol. 43(2), 250-260 (2008)... 

S.S. Chang, D.H. Kang, Alicyclobacillus spp. in the fruit juice industry history, characteristics, and current isolation/detection procedures. Crit. Rev. Microbiol. 30(2), 55-74 (2004)... 

Bartolucci S, De Simone G, Galdiero S, Improta R, Menchise V, Pedone G, Pedone E, Saviano M An integrated structural and computational study of the thermostability of two thioredoxin mutants from Alicyclobacillus acidocaldari-us. J Bacteriol 2003, 185(14) 4285 289. 

Among the prokaryotic organisms, peptidases of seven catalytic types are found. Only one, the glutamic peptidase, has been recently described in prokaryotes in the thermoacidophilic bacteria Alicyclobacillus sp. DSM 15716 (Jensen et al. 2010). In the next section, some peptidases from the different catalytic types will be cited below, but the peptidases fi-om prokaryote can be found in MEROPS database (Rawlings et al. 2010). 

Squalene Cyclization. A bacterial squalene cydase has been firstly characterized in a cell-free system from an Acetobacter species (28). This enzymic activity has been reported later from several other bacteria (29). More recently the enzyme has been isolated from Alicyclobacillus acido-caldarius (30), Rhodopseudomonas palustris (31) and Zymomonas mobi-... 

The syntheses of [5- H] labelled 5-epishikimic acid and [6 H] labelled shikimic acid have been achieved from L-shikimic acid. The synthesis of several deuterated and "C derivatives of shikimic acid has been described in order to study the biosynthesis of ansatrienin (a member of the ansamycin antibiotics). These derivatives were also used in feeding experiments to determine the biosynthesis of the cyclohexanecarboxylic acid starter unit of ca-cyclohexyl fatty acids in Alicyclobacillus acidocaldarius. ... 

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