Biosynthetic enzymes reductases

In order to produce PHAs in plants it is necessary to introduce the biosynthetic enzymes from bacteria. PHB represents the best characterized and simplest form of PHA, and the synthetic pathway (Figure 4.2) has been extensively studied in Ralstonia eutropha. 30,31 Starting from acetyl-CoA, a P-ketothiolase is required in order to form acetoacetyl-CoA. This is then reduced by a NADPH-dependent acetoacetyl-CoA reductase, which gives rise to 3-hydroxybutyryl-CoA. The latter intermediate is the substrate for the polymerization reaction catalyzed by polyhydroxybutyrate synthase.30 In Ralstonia eutropha, the thiolase, reductase, and synthase genes make up an operon.31... 

DFR belongs to the single-domain-reductase/epimerase/dehydrogenase (RED) protein family, which has also been termed the short chain dehydrogenase/reductase (SDR) superfamily. This contains other flavonoid biosynthetic enzymes, in particular the anthocyanidin reductase (ANR), leucoanthocyanidin reductase (EAR), isoflavone reductase (IFR), and vestitone reductase (VR), as well as mammalian, bacterial, and other plant enzymes. ... 

Rationally, 5/3-reduclase deficiency would not be a cause of MPH, but it seems appropriate to place this disorder adjacent to its 5a-counterpart. 5/3-Reductase (AKR1D1) is an essential bile-acid biosynthetic enzyme and patients with disabling mutations in this enzyme have a clinical phenotype associated with cholestasis and fiver failure. In addition to its importance in bile-acid synthesis, this aldoketo-reductase is responsible for reducing approximately two-thirds of the mass of synthesized androgens, corticosteroids, and aldosterone prior to their excretion, so has a vital role in steroid metabolism. 

The active form of folic acid, tetrahydrofolic acid (THF), is produced from folate by dihydrofolate reductase in a two-step reaction requiring two moles of NADPH. The carbon unit carried by THF is bound to nitrogen N5 or N10, or to both N5 and N10. THF allows one-carbon compounds to be recognized and manipulated by biosynthetic enzymes. Figure 20.11 shows the structures of the various members of the THF family, and indicates the sources of the one-carbon units and the synthetic reactions in which the specific members participate. 

The opposite sequence, reduction of nitrate and nitrite ions, provides a major route of acquisition of ammonia for incorporation into cells by bacteria, fungi, and green plants (Fig. 24-1). Assimilatory (biosynthetic) nitrate reductases catalyze the two-electron reduction of nitrate to nitrite (Eq. 16-61). This is thought to occur at the molybdenum atom of the large 900-residue highly regulated793 molybdopterin-dependent enzyme. In green plants the reductant is... 

Mammals must obtain their tetrahydrofolate requirements from their diet, but microorganisms are able to synthesize this material. This offers scope for selective action and led to the use of sulphanilamide and other antibacterial sulpha drugs, compounds which competitively inhibit dihydropteroate synthase, the biosynthetic enzyme incorporating p-aminobenzoic acid into the structure. These sulpha drugs thus act as antimetabolites of p-aminobenzoate. Specific dihydrofolate reductase inhibitors have also become especially useful as antibacterials,... 

The phbA, phbB, and phbC genes from Alcaligenes eutrophus (Ralstonia eutrophus) encoding the biosynthetic enzymes (3-ketothiolase, acetoacetyl-CoA reductase (NADPH-dependent), and PHB synthase, respectively, have been cloned into E. coli (Scheme 19.42).339-342 The use of in vitro evolution using error-prone polymerase chain reaction has led to enhanced accumulation of PHA in a resultant recombinant strain.343 Additional studies to enhance the biosynthesis of PHB through the use of metabolic engineering have been discussed.344... 

Cell line selection is one of the traditional and effective approaches to enhancing metabolite accumulation, and biochemical studies provide the fundamental information for the intentional regulation of secondary metabolism in plant cells. In a carrot suspension culture regulated by 2,4-dichlorophenoxyace-tic acid, Ozeki et al. [7] found that there was a correlation between anthocyanin synthesis and morphological differentiation for somatic embryogenesis they also demonstrated the induction and repression of phenylalanine ammonia lyase (PAL) and chalcone synthase correlated with formation of the respective mRNAs. Two biosynthetic enzymes, i. e., PAL and 3-hydroxymethylglutaryl-CoA reductase, were also related with shikonin formation in Lithospermum erythro-rhizon cultures [8]. 

The provision of reducing equivalents to phenylalanine hydroxylase is dependent on reduction of dihydrobiopterin by NADH catalyzed by the enzyme dihydropteridine reductase, as shown in Figure 38-2. This reduction is dependent on the availability of biopterin and therefore on the biopterin synthetic pathway. Thus any genetic or protein folding defect in either dihydropteridine reductase or the biopterin biosynthetic enzymes would compromise the efficacy of phenylalanine hydroxylation to tyrosine resulting in hyperphenylalaninemia and also phenylketonuria resulting from inaease transamination of phenylalanine to phenylpyruvate. 

A short-term regulation mechanism for cholesterol 7a-hydroxylase activity has been investigated recently in rat liver. The enzyme appears to exist in two forms, which are interconverted by cytosolic fiictors (K12). These foctors may correspond to a protein kinase and a phosphatase, which have been proposed to regulate cholesterol 7a-hydroxylase activity by a phosphorylation (active form)-dephosphorylation (inactive form) mechanism (S9). Another enzyme utilizing cholesterol as substrate, acyl-CoA cholesterol O-acyltransferase (EC 2.3.1.26), may also be regulated in this way, while the biosynthetic enzyme, HMC-CoA reductase, is inhibited in the phosphory-lated form (SIO). Thus, short-term regulation of the concentration of un-esterified cholesterol in the liver may be achieved by coordinate control of these three key enzymes in cholesterol metabolism by reversible phosphorylation (SIO). 

It has also been proposed that hpids, Upid metabolites and cytosolic lipid-binding proteins interact to regulate sterol biosynthesis [199] (see also Chapter 3). Under this model, the accumulation in cells of certain hpids or free hpid intermediates inhibits sterol biosynthetic enzymes. The level of the binding protein for a particular Upid would determine the threshold concentration of Upid needed to produce inhibition [199]. Such a mechanism would allow coordination between the biosynthesis of sterols and other Upids. Fatty acyl-CoAs and Z-protein, the fatty acyl-CoA-binding protein, may modulate HMG-CoA reductase activity [199-201] since physiological... 

Raising the cellular cholesterol content not only stops transcription of the genes encoding the cholesterol biosynthetic enzymes, but also leads to accelerated degradation of the rate-limiting enzyme, HMG-CoA reductase [19]. In cholesterol-depleted cells, HMG-CoA reductase is a stable protein that is degraded slowly (T, = 13 h) (J.R. Faust,... 

Since the PKS (polyketide synthase) gene cluster for actinorhodin (act), an antibiotic produced by Streptomyces coelicolor[ 109], was cloned, more than 20 different gene clusters encoding polyketide biosynthetic enzymes have been isolated from various organisms, mostly actinomycetes, and characterized [98, 100]. Bacterial PKSs are classified into two broad types based on gene organization and biosynthetic mechanisms [98, 100, 102]. In modular PKSs (or type I), discrete multifunctional enzymes control the sequential addition of thioester units and their subsequent modification to produce macrocyclic compounds (or complex polyketides). Type I PKSs are exemplified by 6-deoxyerythronolide B synthase (DEBS), which catalyzes the formation of the macrolactone portion of erythromycin A, an antibiotic produced by Saccharopolyspora erythraea. There are 7 different active-site domains in DEBS, but a given module contains only 3 to 6 active sites. Three domains, acyl carrier protein (ACP), acyltransferase (AT), and P-ketoacyl-ACP synthase (KS), constitute a minimum module. Some modules contain additional domains for reduction of p-carbons, e.g., P-ketoacyl-ACP reductase (KR), dehydratase (DH), and enoyl reductase (ER). The thioesterase-cyclase (TE) protein is present only at the end of module 6. 

S ATP + dephospho-[[hydroxymethylglutaryl-CoA reductase (NADPH)] kinase] <1, 2> (<1,2> phosphorylation activates EC 2.7.1.109 [1-8] <1> involved in regulation cascade of hydroxymethylglutaryl-CoA reductase, EC 1.1.1.34 [2] <1> important for the responses of cells to metabolic stresses such as lack of ceU nutrients, hypoxia, ischemia and muscular exercise [9] <1> bicyclic phosporylation system, enzyme is believed to be involved in protecting cells against ATP depletion due to environmental stress by inactivating several key biosynthetic enzymes [7]) [1-9]... 

Resistance to End Product Repression, a) Antimetabolites. The use of toxic antimetabolites to select resistant cultures often yields mutants whose normal biosynthetic pathway enzymes are not repressed by the end product. For example, trifluoreoleucine selects mutants with derepressed levels (as much as 10-fold) of leucine biosynthetic enzymes (Calvo and Calvo, 1967). Certain canavanine-resistant mutants produce 30 times more of the arginine pathway enzymes than do their sensitive parents (Jacoby and Gorini, 1967). Mutants of Lactobacillus casei resistant to dichloroamethopterin are derepressed 80-fold in their ability to form thymidylate synthetase (Crusberg and Kisliuk, 1969). When Diplococcus pneumoniae mutants are selected on the basis of resistance to amethopterin, these cultures produce 100 times as much dihydrofolate reductase as the parent culture (Sirotnak et al, 1969). 

The use of undifferentiated cultures proved to be unsuccessful for tropane alkaloids production. These alkaloids are produced in normal and transformed roots [42, 51]. Several lines of evidence suggest that the differentiation of the tissue is necessary for the synthesis of these metabolites [42, 51]. Different studies suggest that this is probably related to the localization of key biosynthetic enzymes [6]. Among them, Suzuki et al. [52, 53] demonstrated that the h6h and pmt genes were expressed specifically in root pericycle of Atropa belladonna plants. In addition, Nakajima et al. [54, 55] pointed out that Tropinone reductase enzymes were accumulated in lateral roots of Hyoscyamus niger. 

Fig. 4 Pathways of benzylisoquinoline biosynthesis. A selection of biosynthetic enzymes is notified with their localization in the cytosol (yellow), the ER membrane (red) or in the lumen of cytosolic vesicles (blue). Informations are taken mainly from Facchini and St-Pierre (2005), Bock et al. (2002), own experiments and other references cited in the text. BBE, berberine bridge enzyme CFS, cheilanthifoline synthase CNMT, coclaurine N-methyltransferase COR, codeinone reductase DBOX, dihydrobenzophen-anthridine oxidase MSH, N-methylstylopine 14-hydroxylase NCS, norcoclaurine synthase NMCH, N-methylcoclaurme 3 -hydroxylase 4 OMT, 3 -hydroxy-N-methylcoclaurine 4 -0-methyltransferase 60MT, norcoclaurine 6-O-methyltransferase 70MT, reticuline 7-O-methyltransferase P6H, protopine 6-hydroxylase SAT, salutaridinol-7-O-acetyltransferase SOR, salutaridineiNADPH 7-oxidoreductase STS, stylopine synthase SAS, salutaridine synthase TNMT, tetrahydroprotoberberine cis-N-methyltrans-ferase TYDC, tyrosine decarboxylase CAS, canadine sjmthase SOMT, scoulerine 9-O-methyltransferase STOX, (S)-tetrahydroprotoberberine oxidase...

At the subcellular level, the morphine biosynthetic pathway combines several cytochrome P-450 enzymes, bound to ER membranes (Chou and Kutchan, 1998) with soluble enzymes that reside in ER derived vesicles, e.g. norcoclaurine synthase, or in the cytosol, e.g. codeinone reductase (Zenk, 1994 Facchini and St-Pierre, 2005). The diverse localization of the biosynthetic enzymes requires several transport steps of the intermediates between cytosol, vesicles and vacuoles. Only one of them has been characterized by pioneering experiments (Deus-Neumann and Zenk, 1986) vacuolar vesicles prepared from Fumaria capreolata accumulate (S)-reticulin or (S)-scoulerin via highly specific transporters, that discriminate between (S)- and (R) stereoisomer and exclude other benzylisoquinolines (sanguinarine, protopine, morphine) and alkaloids of unrelated families (e.g. indoles or tropanes). Uptake is energized by the pH gradient across the tonoplast. Accumulated... 

FIGURE 8 The LDL receptor pathway. LDL is internalized via receptor-mediated endocytosis. The endosomes are a sorting compartment the receptor recycles to the plasma membrane, while the LDL is delivered to the lysosomes, where the cholesterol esters are hydrolyzed by lysosomal lipases. The free cholesterol then exits the lysosome and Is able to Inhibit de novo cholesterol synthesis by reducing the abundance of several cholesterol biosynthetic enzymes (e.g., HMG-CoA reductase) and the LDL receptor. Cells protect themselves from cholesterol toxicity by re-esterlfying cholesterol to form a cytoplasmic cholesterol ester droplet. [From Brown, M. S., and Goldstein, J. L. (1986). Science 232, 34-47.]... 

The search for inhibitors of this pathway began with the first key regulatory enzyme, HMG CoA reductase. Several clinically useful inhibitors of HMG CoA reductase are now known. One of the most successful, Mevacor, produced by Merck, is one of the pharmaceutical industry s best selling products. However, the problem with inhibiting a branched biosynthetic pathway at an early point is that the biosynthesis of other crucial biomolecules may also be inhibited. Indeed, there is some evidence that levels of ubiquinone and the dolichols are affected by some HMG CoA reductase inhibitors. Consequently, efforts have recently been directed towards finding inhibitors of squalene synthase, the enzyme controlling the first step on the route to cholesterol after the FPP branch point. 

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