Biosynthesis in vitro

A -Piperideine (17) has been shown to be a precursor of quinolizidine alkaloids in whole plants (cf. Vol. 8, p. 3). However, neither it nor its self-condensation products could be detected as products in the enzymic reaction. [This conclusion is not completely unambiguous, albeit reasonably safe, because the products of the reaction of diamine oxidase, the first of which is (17), were simply compared with those of the alkaloid synthase reaction by g.l.c., and the products of the two reactions were found to be different].11 It seems likely at this stage that (17) is not normally implicated in quinolizidine biosynthesis but can be substituted for an enzyme-generated intermediate via its open form (32) (see Scheme 5). Since no intermediates earlier than (27) could be detected, it is suggested that biosynthesis in vitro and in vivo proceeds by a series of enzyme-linked intermediates (see Scheme 5), none of which is desorbed from the enzyme or enzyme-complex until (27) is liberated. However, in some plants, biosynthesis must stop with the liberation of a compound (31), having the lupinine skeleton... 

Keywords Biocatalysis Biosynthesis In vitro reconstitution Natural products ... 

V. MECHANISTIC INSIGHTS FROM BIOSYNTHESIS IN VITRO OF AROMATIC POLYKETIDES... 

Two other findings from the MCAT experiments have relevance to achieving total biosynthesis in vitro. Investigations into the effects of varying protein concentrations on polyketide production demonstrated that the optimal composition of a minimal PKS is not stoichiometric in each of the components (KS, CLF, and ACP) [30], Increasing the proportion of the ACP relative to the KS/CLF pair caused an enhancement in turnover even at more than 100-fold excess of ACP, there appeared to be no indication of saturation. This result has important implications for the composition of efficient one-pot biosynthetic assemblies. The experiments also illustrated that domains can be usefully engineered to improve yields. In this case, the ACP was modified to prevent its inactivation by formation of an internal disulfide between its active thiol and a remote cysteine. Optimizing... 

To the extent that they are covalently linked, the complex polyketide synthases are less reliant on association for function. In most systems, however, transient docking between multienzymes is required. Recently, it has been demonstrated that the regions of sequence upstream from N-terminal modules and downstream from C-terminal modules (referred to as interpolypeptide linkers) play a crucial role in the assembly of functional modules in vivo [78]. Clearly, the presence of such linkers will also be important for productive biosynthesis in vitro using multiprotein systems. 

Itaya, N.M., Buckeridge, M.S., and Figueiredo-Ribeiro, R.C.L., Biosynthesis in vitro of high-molecular-mass fructan by cell-free extracts from tuberous roots of Viguiera discolor (Asteraceae), New Phytol., 136, 53-60, 1997. 

Three acetylenic compounds (Figure 3) were compared to precocene II (1) for their ability to Inhibit JH biosynthesis in vitro, to inhibit oocyte growth in vivo, and to inhibit midgut microsomal (I.e. extra-allatal) cytochrome P-450 monooxygenases. The compounds were chosen to represent an analog of the substrate of the allatal monooxygenase, methyl farnesoate, with the... 

Inhibition of JH III biosynthesis In vitro. All three acetylenic compounds were significantly better Inhibitors of JH biosynthesis than precocene II under similar Incubation conditions (Fig. 4). Compound 2 was the best Inhibitor tested here, with an I50 of 16 pM, more than 25 times better than precocene II. Whether this better performance as an Inhibitor Is due to a closer structural analogy with the natural substrate of the epoxidase or whether it Is a consequence of the presumed difference In the mode of action (Irreversible Inhibition of the epoxidase for cytotoxicity of precocene epoxide eventually resulting In decreased JH biosynthetic rate for 1) Is not presently known. The Insecticide synergist (4) proved to be about 7 times better than precocene II. Its activity as an inhibitor of JH biosynthesis by D. punctata CA is similar to that of 3 and the methylenedioxyphenyl analog of JH (Ro 20-3600). 

Nakamura, M, Hall, P F, Inhibition by 5-thio-D-glucopyranose of protein biosynthesis in vitro in spermatids from rat testis, Biochim. Biophys. Acta, 447, 474-483, 1976. 

Pratt, O F., and Finney, J.R. (1977 . Chemical inhibitors of juvenile hormone biosynthesis in vitro. In Crop Protection Ajiftvrr.v (Mcbarlane, N.R., ed), pp. II3-132 Academic Press. London. 

Protein biosynthesis is essential for all cells and thus provides another important target. Indeed, a number of alkaloids have been detected (although not too many have been studied in this context) which inhibit protein biosynthesis in vitro. Emetine from Cephaelis ipecacuanha (Rubiaceae) is the most potent plant constituent other alkaloids with the same ability include harringtonine, homoharringtonine, cryptopleurine, tubulosine, hemanthamine, lycorine, narciclasine, pretazettine, pseudolycorine, tylocrepine, and tylopherine [5] and furthermore, ajmaline, berberine, boldine, cinchonine, cinchonidine, harmalin, harmin, lobeline, norharman, papaverine, quinidine, quinine, salsoline, sanguinarine,... 

Usnic Acid. 2I6 Diacetyl 7t9-dihydraxy 8i9b-di-methyl-It3(2H,9bH)-dibenzofurandione usninic acid usnein Usniacin. C H,407 mol wt 344.31. C 62.79%. H 4.68%, O 32.53%. Antibacterial substance found in lichens. Isolation from varieties of Usnea barbota (L.) Wigg., Usneaceae Rochleder, Heidi> Ann. 48, If (1843) Widman Ann. 310, 230 (1900) 324, 139 (1902). Isoin from Ramalina reticulata Marshak, Public Health Reports 62, 3 (1947) Stark et of., J. Am. Chem. Soc. 72, 1819 (1950). Occurs in nature in both the d- and /-forms as well as a racemic mixture. Structure Curd, Robertson, J. Chem. Soc. 1937, 894 Schopf, Ross, Ann. 546, (1941) Barton, Brunn, /. Chem. Soc. 1953, 603. Resolution of ( )-usnic acid Dean et ai, ibid. 1250. Synthesis Barton et a/., ibid. 1956, 530. Biosynthesis in vitro Penttila, FaJes, Chem. Commun 1966, 656. Abs config of (+)-fomv S. Huneck et aL, Tetrahedron Letters 22, 351 (1981). 

Haidar, S., P.B. Ehmer, S. Barassin, C. Batzl-Hartmann, and R.W. Hartmann (2003). Effects of novel 17alpha-hydroxylase/C 17,20-lyase (P450 17, CYP 17) inhibitors on androgen biosynthesis in vitro and in vivo. J. Steroid Biochem. Mol. Biol. 84, 555-562. 

Camara, B. and R. Moneger, Carotenoid biosynthesis. In vitro conversion of antheraxanthin to capsanthin by a chromoplast enriched fraction of Capsicum fruits, Biochem. Biophys. Res. Commun., 99, 1117-1122 (1981). 

Wouters W, De Coster R, Van Dun J, Krekels MDWG, Dillen A, Raeymaekers A, Freyne E, Van Gelder J, Sanz G, Venet M, Janssen M (1990) Comparative effects of the aromatase inhibitor R76713 and of its enantiomers R83839 and R83842 on steroid biosynthesis in vitro and in vivo. J Steroid Biochem Mol Biol 37 1049-1054... 

Sundberg, D. K., Bennett, B., Wendel, O. T., and Morris, M., 1980, Hypothalamic catecholamine biosynthesis in vitro as measured by liquid chromatography and electrochemical detection, Res. Commun. Chem. Pathol. Pharmacol. 29 599-602. 

Experiments made on pyridoxal-deficient animals suggested that pyridoxal phosphate and iron are required for the biosynthesis of porphyrin. It was later established that pyridoxal phosphate is required for the formation of -amino levulinic acid, probably by participating in the formation of active glycine. The condensation of succinate and glycine leads to the formation of a very labile a-amino-j -keto adipic acid. The participation of a-amino-jS-keto adipic acid as an intermediate in the reaction was established in experiments proving the acid to be an efficient precursor of porphyrin biosynthesis in vitro. An enzyme system capable of catalyzing the succinyl CoA-glycine condensation and the decarboxylation of the intermediate to yield amino levulinic acid has also been obtained from a particular fraction of chicken erythrocyte. In liver, an enzyme has been found in the mitochondria [132]. 

Kellmaim R, Neilan BA (2007) Biochemical characterisatimi of paralytic shellfish toxin biosynthesis in vitro. J Phycol 43 497-508... 

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