Purified Enzyme Systems

The 3-ketothiolase has been purified and investigated from several poly(3HB)-synthesizing bacteria including Azotobacter beijerinckii [10], Ral-stonia eutropha [11], Zoogloea ramigera [12], Rhodococcus ruber [13], and Methylobacterium rhodesianum [14]. In R. eutropha the 3-ketothiolase occurs in two different forms, called A and B, which have different substrate specificities [11,15]. In the thiolytic reaction, enzyme A is only active with C4 and C5 3-ketoacyl-CoA whereas the substrate spectrum of enzyme B is much broader, since it is active with C4 to C10 substrates [11]. Enzyme A seems to be the main biosynthetic enzyme acting in the poly(3HB) synthesis pathway, while enzyme B should rather have a catabolic function in fatty-acid metabolism. However, in vitro studies with reconstituted purified enzyme systems have demonstrated that enzyme B can also contribute to poly(3HB) synthesis [15]. 

This review of the biosynthesis of the bisindole alkaloids of C. roseus is organized along a developing biosynthetic pathway, as far as possible, and relies on the notion that the most sophisticated studies are those utilizing the purified enzyme systems. Biosynthetic studies on the other monoterpene indole alkaloids are not reviewed here. 

The unsaturated Cg aldehydes and alcohols probably arise in wheat from cleavage of the 9—10 double bond of unsaturated C g fatty acids, linoleic and linolenic acids (Figure 2). Galliard and coworkers (12,13) have partially purified enzyme systems capable of catalyzing these transformations. Lipoxygenase initially converts linoleic and linolenic acids to 9-hydroperoxides which are subsequently cleaved by hydroperoxide lyase to volatile Cg unsaturated aldehydes and 9-oxo-nonanoic acid. The 3-enals are the primary volatile cleavage products from the fatty acids and these are transformed by an isomerase to the more stable 2—enals (14). The 3—enals are rather unstable but Hatanaka et al. (15) have confirmed their presence in plant tissue with authentic samples. The Cg... 

These results show then that in the introduction of the first hydroxy-group on an aromatic substrate, hydrogen loss after 1,2-migration is stereospecific rather than isotopically controlled and it is the migrating hydrogen which is retained. Lack of the appropriate enzyme(s) in the previous experiments (which were with purified or partially purified enzyme systems) would account for results differing from those found in the intact plant. These results were accommodated within a plausible sequence (Scheme 11) which finds in vitro support. The intermediate (109) may be included, provided hydrogen loss is enzymically controlled and thereby stereospecific. 

Neither El nor E2, together or alone, mediated the formation of acrylyl- or lactyl-CoA from the respective acid plus CoA and acetyl phosphate or acetyl-CoA. However, a CoA transferase that catalyzes the formation of the CoA thioesters of lactate, acrylate, and propionate from acetyl-CoA has been isolated from C. propionicum (233). It is curious that acrylyl-CoA could not be isolated as a product with the crude or purified enzyme system, although acrylyl-CoA is readily converted to lactyl-CoA in the purified system. Abeles suggested that perhaps acrylyl-CoA exists as an enzyme-bound intermediate that requires the presence of a reductase for release as propionate. In this manner the organism is protected from buildup of toxic amounts of any acrylyl intermediates, which are known to undergo Michael addition reactions with biological nucleophiles. 

The product, arbutin, was identified by chromatographic and colorimetric methods. A large variety of di- and tri-phenols (but no monophenols) could substitute for hydroquinones as acceptors in the purified-enzyme system presumably, the enzymes using monophenols as substrates had been lost during purification. 

Quantitative Reaction Phenotyping Expressed or Purified Enzyme Systems... 

In summary, purified enzyme systems and synthetic enzymatic pathways are opening up new opportunities for high-yielding bioconversions. It is clear that... 

Crude cell lysates offer an alternative approach to purifying individual enzymes to create a synthetic reaction network. A key difference is that native enzymes in the lysate can enable ATP and cofactor regeneration [13, 71]. While this provides a benefit when compared to purified enzyme systems, the cost adds to the complexity. The emergence of crude extract-based CFME is a new development only in recent years. 

The feedback inhibition of the first specific enzyme of purine biosynthesis, amidophos-phoribosyltransferase, by the purine ribonucleotides AMP and GMP was described in a partially purified enzyme system over 20 years ago(l). The molecular basis for this inhibition, namely the aggregation of active subunits to an enzymatically inactive larger form in the presence of purine ribonucleotides, was described with the amidophosphoribosyl-transferase obtained from human placenta seven years ago(2). Phosphoribosylpyrophos-phate (PRPP) activates the mammalian enzyme by causing disaggregation of the inhibited large form to the catalytically active small form. 

Purified CYP2C13 exhibits high testosterone hydroxylase activity in a purified enzyme system, but this enzyme makes only marginal contributions to liver microsomal testosterone hydroxylation [323]... 

Some of these steps can be bypassed in nonphos-phorylating particles, and the spectrophotometric methods do not exclude the possibility that some of the components that seem to participate in the electron transport chain are only parasites. Definite information on these mechanisms will be obtained only when in vitro reconstruction with purified enzyme systems is possible. 

FAD is similar in structure to DPN. It differs in that riboflavin replaces the nicotinamide riboside moiety. It is formed when red blood cells are incubated with riboflavin.A purified enzyme system from yeast catalyzes the synthesis of this coenzyme from ATP and flavin mononucleotide. ... 

Evidence was obtained that fatty acid synthesis proceeds in the cytoplasmic fraction rather than in the mitochondria. A supernatant enzyme system was able to synthesize palmitic acid starting from acetyl-CoA in the presence of NADPH, Mn++, HCOg- and ATP. The purified enzyme system was free of oxidation enzymes. HCO3" was not incorporated into the fatty acid but had a cofactor role. Subsequent experiments showed that acetyl-CoA is carboxylated to malonyl-CoA by acetyl-CoA-carboxylase, which contains d-biotin as coenzyme. Early investigations gave strong evidence for the participation of biotin in a number of carboxylation (COg-fixation) reactions (Lardy et al. 1956). The enzyme acetyl-CoA-carboxylase has been isolated (Brady et al. 1958, Lynen et al. 1959, Wakil et al. 1958). COg forms the free carboxy-group of malonyl-CoA. The equation of the malonyl-CoA synthesis is ... 

Although it has not yet been possible to study the synthesis of proteins in purified enzyme systems, a considerable number of model reactions which involve the formation of peptide or amide linkages have been investigated. These include the synthesis of glutamine, hippuric acid, acetylated amines, glutathione, and urea. It is of significance that every one of these model reactions, studied in isolated enzyme systems, has been shown to require ATP. In addition, two of these reactions, the formation of hippuric acid and the acetylation of amines, have been shown to require coenzyme A. 

The studies of Nakada and Sund (38) indicate that the oxidation of glyoxylic acid to formate and COx is not a simple reaction. Employing a partially purified enzyme system from rat liver mitochondria these authors observed that addition of L-glutamate and DPN were required for the decarboxylation. In addition the velocity of the reaction was enhanced by certain divalent cations, particularly Mn and thiamine pyropho hate. Glutamate was most efficient when present in a concentration equimolar with yoxylate. 

In the purified enzyme system ascorbic acid becomes progressively less effective while 2,6-dichlbrophenolindophenol gains in efficiency 211). These results suggest that the role of ascorbic acid and other effective compounds is to maintain some particular reactant of the enzyme system in a reduced state. 

Analogs of the natural deoxynucleoside triphosphates can be used for DNA synthesis, both in vivo and in vitro, but substitution of unnatural for natural deoxynucleotide must conform with the requirements for base-pairing in the Watson-Crick model of DNA. Thus with the purified enzyme system, thymine could be replaced by uracil or 5-bromouracil, 5-methyl- and 5-bromocytosine for cytosine, and hypoxanthine for guanine ( 6). Although chemically synthesized deoxyuridine triphosphate can be incorporated into DNA, there is apparently no kinase in nature which phosphorylates deoxyuridylate to the triphosphate stage. This may account for the absence of uracil nucleotides in DNA ( 6). 

---