Biosynthetic enzymes

MS-based substrate specificity assays that are applied to NRPS domains are mainly aimed to characterize native and alternative biosynthetic substrates. Table 1 presents biosynthetic substrate pools for substrate screening of catalytic NRPS domains and tailoring enzymes. Biosynthetic substrate pools are specified for the two purposes of assaying NRPS substrate specificity as mentioned above (a) substrate identification and (b) determination of substrate tolerance. The biosynthetic substrate pools for each specific domain and for each experimental purpose are explained as follows. 

Cahoon, E.B. K.G. Ripp S.E. Hall A.J. Kinney. Formation of conjugated delta 8, delta 10 double bonds by delta 12-oleic acid desaturase related enzymes biosynthetic origin of calendic acid./. Biol Chem. 2001,276, 2637-2643. 

Phenolic compounds are commonplace natural products Figure 24 2 presents a sampling of some naturally occurring phenols Phenolic natural products can arise by a number of different biosynthetic pathways In animals aromatic rings are hydroxylated by way of arene oxide intermediates formed by the enzyme catalyzed reaction between an aromatic ring and molecular oxygen... 

Before leaving this biosynthetic scheme notice that PGE2 has four chirality cen ters Even though arachidomc acid is achiral only the stereoisomer shown m the equa tion IS formed Moreover it is formed as a single enantiomer The stereochemistry is controlled by the interaction of the substrate with the enzymes that act on it Enzymes offer a chiral environment m which biochemical transformations occur and enzyme catalyzed reactions almost always lead to a single stereoisomer Many more examples will be seen m this chapter... 

FIGURE 27 5 Tyrosine is the biosynthetic precursor to a number of neurotransmit ters Each transformation IS enzyme catalyzed Hydroxy lation of the aromatic ring of tyrosine converts it to 3 4 dihyd roxyphenylalanine (l dopa) decarboxylation of which gives dopamine Hy droxylation of the benzylic carbon of dopamine con verts It to norepinephrine (noradrenaline) and methy lation of the ammo group of norepinephrine yields epi nephrine (adrenaline)... 

Molybdate is also known as an inhibitor of the important enzyme ATP sulfurylase where ATP is adenosine triphosphate, which activates sulfate for participation in biosynthetic pathways (56). The tetrahedral molybdate dianion, MoO , substitutes for the tetrahedral sulfate dianion, SO , and leads to futile cycling of the enzyme and total inhibition of sulfate activation. Molybdate is also a co-effector in the receptor for steroids (qv) in mammalian systems, a biochemical finding that may also have physiological implications (57). 

One of the most exciting discoveries related to quinone/hydroquinone chemistry is thek synthesis by biosynthetic routes (12,13). Using bacterial enzymes to convert D-glucose [50-99-7] (7) to either 1,2- or l,4-ben2enediol allows the use of renewable raw material to replace traditional petrochemicals. The promise of reduced dependence on caustic solutions and the use of transition-metal catalysts for thek synthesis are attractive in spite of the scientific and economic problems still to be solved. 

Vitamins are classified by their solubiUty characteristics iato fat-soluble and water-soluble groups. The fat-soluble vitamins A, E, and K result from the isoprenoid biosynthetic pathway. Vitamin A is derived by enzymic cleavage of the symmetrical C q beta-carotene, also known as pro-vitamin A. Vitamins E and K result from condensations of phytyldiphosphate (C2q) with aromatic components derived from shikimic acid. Vitamin D results from photochemical ring opening of 7-dehydrocholesterol, itself derived from squalene (C q). 

Over 250 analogues of the B vitamers have been reported (11,100). Nearly all have low vitamin B activity and some show antagonism. Among these are the 4-deshydroxy analogue, pyridoxine 4-ethers, and 4-amino-5-hydroxymeth5i-2-methyipyrimidine, a biosynthetic precursor to thiamine. StmcturaHy unrelated antagonists include dmgs such as isoniazid, cycloserine, and penicillamine, which are known to bind to pyridoxal enzyme active sites (4). 

The biosynthetic sequence for other aerobic bacteria appears, where known, to be similar to that in Pseudomonas dentrificans although the genes involved, and thus the enzymes, exhibit differences. 

The proposed pathway for the biosynthesis of the avermectins (Fig. 3) has been described in a review (23). Some of the details are yet to be elucidated, although the steps, in general, are based on firm evidence from four types of studies incorporation of labeled precursors, conversion of putative intermediates by producing strains and blocked mutants, in vitro measurement of biosynthetic enzymes, and studies with enzyme inhibitors. The biosynthesis of the oleandrose units was elucidated from studies using and labeled glucose, which indicated a direct conversion of glucose to... 

One of the most interesting uses for cinnamic acid in recent years has been as a raw material in the preparation of L-phenylalanine [63-91-2] the key intermediate for the synthetic dipeptide sweetener aspartame (25). Genex has described a biosynthetic route to L-phenylalanine which involves treatment of immobilized ceUs of R rubra containing the enzyme phenylalanine ammonia lyase (PAT,) with ammonium cinnamate [25459-05-6] (26). 

Fig. 2. Biosynthetic pathway for epinephrine, norepinephrine, and dopamine. The enzymes cataly2ing the reaction are (1) tyrosine hydroxylase (TH), tetrahydrobiopterin and O2 are also involved (2) dopa decarboxylase (DDC) with pyridoxal phosphate (3) dopamine-P-oxidase (DBH) with ascorbate, O2 in the adrenal medulla, brain, and peripheral nerves and (4) phenethanolamine A/-methyltransferase (PNMT) with. Cadenosylmethionine in the adrenal...

FIGURE 26.10 The biosynthetic conversion of squa-lene to cholesterol proceeds through lanosterol. Lano-sterol is formed by enzyme-catalyzed cyclization of the 2,3-epoxide of squalene. 

Nicotinamide is an essential part of two important coenzymes nicotinamide adenine dinucleotide (NAD ) and nicotinamide adenine dinucleotide phosphate (NADP ) (Figure 18.19). The reduced forms of these coenzymes are NADH and NADPH. The nieotinamide eoenzymes (also known as pyridine nucleotides) are electron carriers. They play vital roles in a variety of enzyme-catalyzed oxidation-reduction reactions. (NAD is an electron acceptor in oxidative (catabolic) pathways and NADPH is an electron donor in reductive (biosynthetic) pathways.) These reactions involve direct transfer of hydride anion either to NAD(P) or from NAD(P)H. The enzymes that facilitate such... 

In animals, the enzymes of fatty acid synthesis are components of one long polypeptide chain, the fatty acid synthase, whereas no similar association exists for the degradative enzymes. (Plants and bacteria employ separate enzymes to carry out the biosynthetic reactions.)... 

Analogous side-chain oxidations occur in various biosynthetic pathways. The neurotransmitter norepinephrine, for instance, is biosynthesized from dopamine by a benzylic hydroxylation reaction. The process is catalyzed by the copper-containing enzyme dopamine /3-monooxygenase and occurs by a radical mechanism. A copper-oxygen species in the enzyme first abstracts the pro-R benzylic hydrogen to give a radical, and a hydroxyl is then transferred from copper to carbon. 

The biomimetic approach to total synthesis draws inspiration from the enzyme-catalyzed conversion of squalene oxide (2) to lanosterol (3) (through polyolefinic cyclization and subsequent rearrangement), a biosynthetic precursor of cholesterol, and the related conversion of squalene oxide (2) to the plant triterpenoid dammaradienol (4) (see Scheme la).3 The dramatic productivity of these enzyme-mediated transformations is obvious in one impressive step, squalene oxide (2), a molecule harboring only a single asymmetric carbon atom, is converted into a stereochemically complex polycyclic framework in a manner that is stereospecific. In both cases, four carbocyclic rings are created at the expense of a single oxirane ring. 

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