Cinnamoyl enzyme

The serine proteases act by forming and hydrolyzing an ester on a serine residue. This was initially established using the nerve gas diisopropyl fluorophosphate, which inactivates serine proteases as well as acetylcholinesterase. It is a very potent inhibitor (it essentially binds in a 1 1 stoichiometry and thus can be used to titrate the active sites) and is extremely toxic in even low amounts. Careful acid or enzymatic hydrolysis (see Section 9.3.6.) of the inactivated enzyme yielded O-phosphoserine, and the serine was identified as residue 195 in the sequence. Chy-motrypsin acts on the compound cinnamoylimidazole, producing an acyl intermediate called cinnamoyl-enzyme which hydrolyzes slowly. This fact was exploited in an active-site titration (see Section 9.2.5.). Cinnamoyl-CT features a spectrum similar to that of the model compound O-cinnamoylserine, on denaturation of the enzyme in urea the spectrum was identical to that of O-acetylserine. Serine proteases act on both esters and amides. 

The pure enzyme was tested for activity against several methylated phenolic and cinnamic acids (Table 2). The enzyme was active on methyl esters of cinnamic acids caffeio p-coumaric> ferulic, and is therefore termed a cinnamoyl esterase (CinnAE). 

Since the imidazolide method proceeds almost quantitatively, it has been used for the synthesis of isotopically labeled esters (see also Section 3.2), and it is always useful for the esterification of sensitive carboxylic acids, alcohols, and phenols under mild conditions. This advantage has been utilized in biochemistry for the study of transacylating enzymes. A number of enzymatic transacylations (e.g., those catalyzed by oc-chymo-trypsin) have been shown to proceed in two steps an acyl group is first transferred from the substrate to the enzyme to form an acyl enzyme, which is then deacylated in a second step. In this context it has been shown[21] that oc-chymotrypsin is rapidly and quantitatively acylated by Af-fraw.s-cinnamoylimidazole to give /ra/w-cinnamoyl-a-chymotrypsin, which can be isolated in preparative quantities and retains its enzymatic activity (see also Chapter 6). 

Members of the CHS/STS family of condensing enzymes are relatively modest-sized proteins of 40-47 kDa that function as homodimers. Each enzyme typically reacts with a cinnamoyl-CoA starter unit and catalyzes three successive chain extensions with reactive acetyl groups derived from enzyme catalyzed decarboxylation of malonyl-CoA.11 Release of the resultant tetraketide together with or prior to polyketide chain cyclization and/or decarboxylation yields chalcone or resveratrol (a stilbene). Notably, CHS and STS catalyze identical reactions up to the formation of the intermediate tetraketide. Divergence occurs during the termination step of the biosynthetic cascade as each tetraketide intermediate undergoes a distinct cyclization reaction (Fig. 12.2). 

The first STS structure solved was that of a pinosylvin-forming STS from Pinus sylvestris. Pine trees can by-pass the C4H reaction and directly produce the CoA thioester of cinnamate that allows this STS to utilize a non-substituted cinnamoyl-CoA starter in vz vo.11 However, when presented in vitro with p-coumaroyl-CoA, the enzyme proves to be comparable in activity to STS enzymes from organisms that utilize the para-substituted cinnamoyl starter. 

All of this evidence supports the existence of tetrahedral intermediates in a-chymotrypsin-catalysed reactions, but it should be noted that O-exchange with water is not observed in deacylation of cinnamoyl- 0-chymotrypsin, in contrast with the hydrolysis of O-cinnamoyl-N-acetylserinamide where such exchange is detected (Bender and Heck, 1967). Lack of exchange in the enzyme reaction could reflect interactions of the tetrahedral intermediate with the protein. 

This enzyme [EC 6.2.1.12] (also referred to as hydroxy-cinnamoyl-CoA ligase and 4-coumarate CoA hgase) catalyzes the reaction of 4-coumarate with ATP and coenzyme A to yield 4-coumaroyl-CoA, AMP, and pyrophosphate (or, diphosphate). 

CHS carries out a series of sequential decarboxylation and condensation reactions, using 4-courmaroyl-CoA (in most species) and three molecules of malonyl-CoA, to produce a poly-ketide intermediate that then undergoes cyclization and aromatization reactions that form the A-ring and the resultant chalcone structure. The chalcone formed from 4-courmaroyl-CoA is naringenin chalcone. However, enzyme preparations and recombinant CHS proteins from some species have been shown to accept other HCA-CoA esters as substrates, such as cinnamoyl-CoA (see, e.g., Ref. 37). In particular, the Hordeum vulgare (barley) CHS2 cDNA encodes a CHS protein that converts feruloyl-CoA and caffeoyl-CoA at the highest rate, and cinnamoyl-CoA and 4-courmaroyl-CoA at lower rates. 

Dugelay, I. et al.. Role of cinnamoyl esterase activities from enzyme preparations on the formation of volatile phenols during winemaking. J. Agric. Food Chem. 41, 2092, 1993. 

The reduction of />coumaroyl-CoA (3.31) to />coumaryl aldehyde (3.69) is catalyzed by the enzyme cinnamoyl-CoA NADP oxidoreductase (CCR). This enzyme was initially purified from soybean cultures (Wegenmayer et al., 1976), and was later on efficiently isolated from lignifying cambium of eucalyps (Eucalyptus gunnii) (Gofifiier et al., 1994). A CCR cDNA was identified in a cDNA library that was screened with oligonucleotiede derived from the peptide sequence of the CCR protein. CCR is considered the first enzyme committed towards the biosynthesis of monolignols and shows... 

The substitution of the phenyl ring necessary for the biosynthesis of coniferyl alcohol (3.79) and sinapyl alcohol (3.81) begins with the hydroxylation of C3. This is a conversion that requires the formation of the ester of /5-coumaroyl-CoA with D-quinate (3.73) or shikimate (3.74) catalyzed by the enzyme hydroxycinnamoyl-CoA shikimate/quinate hydroxy-cinnamoyl transferase (HCT Hoffmann et al., 2003). The hydroxylation of this ester intermediate is catalyzed by the enzyme /i-coumarovl-Co A 3 -hydroxylase (C3 H Schoch et al., 2001 Franke et al., 2002a,b). The resulting shikimate or quinate ester (3.75 3.76) is subsequently hydrolyzed by the same HCT, resulting in caffeoyl-CoA (3.36). 

Figure 3-9. Biosynthesis of monolignols. The enzymes involved in this pathway are ( ) hydroxycinnamoyl-CoA shikimate/quinate hydroxy-cinnamoyl transferase, (b) p-coumaroyl-CoA 3 -hydroxylase (E.C. 1.14.14.1), (c) caffeoyl-CoA O-methy 1 Iranslerasc (E.C. 2.1.1.104), (d) cinnamoyl-CoA reductase (E.C. 1.2.1.44) (e) cinnamyl alcohol dehydrogenase (E.C. 1.1.1.195), (f) coniferyl aldehyde/coniferyl alcohol 5-hydroxylase (E.C. 1.14.13), (g) coniferaldehyde/coniferyl alcohol O-methyltransferase (E.C. 2.1.1.68).

Lacombe, E., Hawkins, S., Van Doorsselaere, J., Piquemal, J., Goffher, D., Poeydomenge, O., Boudet, A.-M., and Grima-Pettenati, J., 1997, Cinnamoyl Co A reductase, the first committed enzyme of the lignin branch biosynthetic pathway cloning, expression and phylogenetic relationships, Plant J. 11 429-441. 

Wegenmayer, H., Ebel, J. and Grisebach, H., 1976, Enzymic synthesis of lignin precursors Purification and properties of a cinnamoyl-CoA NADPH reductase from cell suspension cultures of soybean (Glycine max L.), Eur. J. Biochem. 65 529-536. 

The polyketide synthases responsible for chain extension of cinnamoyl-CoA starter units leading to flavonoids and stilbenes, and of anthraniloyl-CoA leading to quinoline and acridine alkaloids (see page 377) do not fall into either of the above categories and have now been termed Type TTT PKSs. These enzymes differ from the other examples in that they are homodimeric proteins, they utilize coenzyme A esters rather than acyl carrier proteins, and they employ a single active site to perform a series of decarboxylation, condensation, cyclization, and aromatization reactions. 

Both structures nicely illustrate the different characteristic oxygenation patterns in aromatic rings derived from the acetate or shikimate pathways. With the stilbenes, it is noted that the terminal ester function is no longer present, and therefore hydrolysis and decarboxylation have also taken place during this transformation. No intermediates, e.g. carboxylated stilbenes, have been detected, and the transformation from cinnamoyl-CoA/malonyl-CoA to stilbene is catalysed by the single enzyme. Resveratrol has assumed greater relevance in recent years as a constituent of grapes and wine, as well as other food products, with antioxidant, anti-inflammatory, anti-platelet, and cancer preventative properties. Coupled with... 

Detection of the intermediate is possible if it has a spectrum sufficiently different from that of the enzyme. The cinnamoyl chymotrypsin intermediate is characterised by a UV maximum at 292 nm the acyl papain intermediate JV-benzoylaminothionacetyl papain has a UV maximum at 313 nm. The UV absorptions of the reactions catalysed by papain and chymotrypsin wax and wane in the presence of substrate giving rise to these intermediates. 

Koenigs, P.M., Faust, B.C. and Porter, N. A. (1993) Photochemistry of enzyme-bound cinnamoyl derivatives. Journal of the American Chemical Society, 115, 9371-9379. 

The extension of a cinnamoyl-CoA chain (126) with malonate leads to the polyke-tide 127, which may be folded differently, depending on the enzymes present in the plant (Fig. 32). Stilbene synthase, for example, converts the polyketide into a stilbene (171) through an aldol-type condensation (86). 

Dugelay, I., Giinata, Z., Bitteur, S., Sapis, J.C., Baumes, R., Bayonove, C. (1992a). Formation of volatile phenols from cinnamic precursors during wine making the role of cinnamoyl esterase from commercial enzymic preparations. In P. Schreier P. Winterhalter (Eds.), Progress in flavour studies (pp. 189-193). Carol Stream Ils. Allured Publishing Co. 

Aspergillus niger Mould for commercial enzyme production (pectinase, hemicellulase) Active cinnamoyl esterase releasing free hydroxycinnamic acids in juices Dugelay et al. (1993)... 

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