A-hydroxylases

Although it is being found that vitamin D metaboUtes play a role ia many different biological functions, metaboHsm primarily occurs to maintain the calcium homeostasis of the body. When calcium semm levels fall below the normal range, 1 a,25-dihydroxy-vitainin is made when calcium levels are at or above this level, 24,25-dihydroxycholecalciferol is made, and 1 a-hydroxylase activity is discontiaued. The calcium homeostasis mechanism iavolves a hypocalcemic stimulus, which iaduces the secretion of parathyroid hormone. This causes phosphate diuresis ia the kidney, which stimulates the 1 a-hydroxylase activity and causes the hydroxylation of 25-hydroxy-vitamin D to 1 a,25-dihydroxycholecalciferol. Parathyroid hormone and 1,25-dihydroxycholecalciferol act at the bone site cooperatively to stimulate calcium mobilization from the bone (see Hormones). Calcium blood levels are also iafluenced by the effects of the metaboUte on intestinal absorption and renal resorption. 

Although essential amino acids are requited by both host and tumor, deprivation of select essential amino acids for 2—3 weeks is tolerated by the host yet exerts a pronounced antiproliferative effect on the tumor. Thus, treatment of mice with indole-3-alkane-a-hydroxylase [63363-76-8] from Pseudomonas, which transforms L-tryptophan [73-22-3] to 3-indolylglycaldehyde, lowers the concentration of L-tryptophan in plasma, brain, and lungs, and inhibits the growth of a variety of tumors (32—34). 

Although /3-oxidation is universally important, there are some instances in which it cannot operate effectively. For example, branched-chain fatty acids with alkyl branches at odd-numbered carbons are not effective substrates for /3-oxidation. For such species, a-oxidation is a useful alternative. Consider phy-tol, a breakdown product of chlorophyll that occurs in the fat of ruminant animals such as sheep and cows and also in dairy products. Ruminants oxidize phytol to phytanic acid, and digestion of phytanic acid in dairy products is thus an important dietary consideration for humans. The methyl group at C-3 will block /3-oxidation, but, as shown in Figure 24.26, phytanic acid a-hydroxylase places an —OFI group at the a-carbon, and phytanic acid a-oxidase decar-boxylates it to yield pristanie add. The CoA ester of this metabolite can undergo /3-oxidation in the normal manner. The terminal product, isobutyryl-CoA, can be sent into the TCA cycle by conversion to succinyl-CoA. 

The hepatic microsomal a-hydroxylase activity for NPYR is inducible in rats by pretreatment with Aroclor, and in hamsters by pretreatment with Aroclor, 3-methylcholanthrene, phenobarbi-tal, and ethanol (10,15,19). In contrast, pretreatment of rats with 3-methylcholanthrene or phenobarbital causes no change or a slight decrease in microsomal NPYR o-hydroxylase activity (19). 

The metabolism of 4-hydroxybenzoate involves conversion into 3,4-dihydroxybenzoate by a hydroxylase that has been purified and characterized from a strain of Pseudomonas... 

Kitcher JO, PW Trudgill, JS Rees (1972) Purification and properties of 2-furoyl-coenzyme A hydroxylase from Pseudomonas putida F2. Biochem J 130 121-132. 

Stimulating activation of vitamin D by 1-a-hydroxylase to cal-citriol (1,25-dihydroxyvitmin D3) to promote calcium absorption in the GI tract and increased calcium mobilization from bone... 

The precursor, 7-dehydrocholesterol is converted by a non-enzymatic reaction to cholecalciferol (calciol). This reaction occurs in skin exposed to sunlight due to irradiation by UV-B light at a wavelength of about 300 nm. Cholecalciferol is transported via carrier proteins to the liver where hydroxylation at carbon-25 occurs in a reaction catalysed by a microsomal cytochrome P450 hydroxylase to form calcidiol. This compound travels to the kidney attached to specific binding proteins, where another cytochrome P450 enzyme, mitochondrial 1-a-hydroxylase, introduces a second hydroxyl group in to the molecule to form the active calcitriol. 

In addition to 1-a-hydroxylase, the kidney also possesses a 24-hydroxylase which uses calcidiol as substrate the product of the reaction, 24,25 dihydroxy D3, is biologically inactive. This represents an important control point in the pathway. The activity of the 1-a-hydroxylase is promoted by calcium ions and the action of PTH acting via a G-protein/cAMP cascade. However, calcitriol itself simultaneously induces the 24-hydroxylase and suppresses 1-a-hydroxylase creating an effective feedback loop (Figure 8.12). 

Figure 1.1 The classic pathway for the conversion of cholesterol into the primary bile acids CA and CDCA, involving the 7 a-hydroxylase enzyme (also known as CYP7A1). Simplified from Dr John Chiang/ The 7 OH group is highlighted with the shaded circle. This group is cleaved to produce the secondary BAs DCA and LCA.

Bile salts are exclusively synthesized in the liver (see A). The slowest step in their biosynthesis is hydroxylation at position 7 by a 7-a-hydroxylase. Cholic acid and other bile acids inhibit this reaction (end-product inhibition). In this way, the bile acids present in the liver regulate the rate of cholesterol utilization. 

The lowered concentration of bile acids returning to the liver by the enterohepatic circulation results in derepression of 7-a-hydroxylase, the rate-limiting enzyme for conversion of cholesterol to bile acids. This results in increased use of cholesterol to replace the excreted bile acids and lowering of hepatic cholesterol (mechanism VI in Fig. 23.2). Thus, similar to the statins, the ultimate actions of the bile acid-sequestering resins are up-regulation of transcription of the LDL receptor gene, increased hepatic receptor activity, and lowering of plasma LDL cholesterol (mechanism VII in Fig. 23.2). 

Franssen et al. [24] pointed out an alternative method of production of nootkatone from valencene catalysed by (-i-)-germacrene A hydroxylase, an enzyme of the cytochrome P450 monooxygenase type that was isolated from chicory roots. In general, this enzyme appeared to accept a broad range of sesquiterpenes and hydroxylates exclusively at the side-chain s isopropenyl group. Valencene is an exception it was not hydroxylated at the side chain, but -nootkatol was formed in the first step (Scheme 22.5) it is not yet clear if the second step is enzyme-catalysed. 

Scheme 22.5 Production of nootkatone from valencene catalysed by (+)-germacrene A hydroxylase [81]...

The multiprotein complex methane monooxygenase (MMO) serves meth-anotrophs to convert methane to methanol. It can be either soluble (sMMO) or membrane bound ( particulate , pMMO) and it typically consists of three components, a reductase (MMOR), a component termed protein B (MMOB) and a hydroxylase denoted MMOH. The nature of the metal cofactors in the latter component are reasonably well understood for sMMO as will be discussed in the non-heme iron section. For the pMMO of Methylococcus capsulatus an obligate requirement for copper was shown. As reported in reference 1 a trinuclear Cu(II) cluster was discussed128 but the number and coordination of coppers still is a matter of continuing investigation since then. 

In the rat, development to adult levels of activity takes about 30 days after which levels decline toward old age. In humans, however, hydroxylase activity increases up to the age of 6 years, reaching levels greater than those in the adult, which only decrease after sexual maturation. Thus the elimination of antipyrine and theophylline was found to be greater in children than in adults. It should be noted, however, that proportions of isoenzymes may be very different in neonates from the adult animal, and the development of the isoenzymes may be different. Thus, in the rat there seem to be four types of development for phase 1 metabolizing enzymes linear increase from birth to adulthood, type A (aniline 4-hydroxylation) low levels until weaning, then an increase to adult levels, type B (N-demethylation) rapid development after birth followed by rapid decline to low levels in adulthood, type C (hydroxylation of 4-methylcoumarin) and rapid increase after birth to a maximum and then decline to adult levels, type D. Patterns of development may be different between sexes as well as between species. For example, in the rat, steroid 16-a-hydroxylase activity toward androst-4-ene-3,17-dione develops in type B fashion in both males and females, but in females, activity starts to disappear at 30 days of age and is undetectable by 40 days. It seems that the monooxygenase system develops largely as a unit, with the rate dependent on species and sex of the animal and the particular substrate. 

The ability of Pseudomonas putida to grow onoctanol is due to two alkane-inducible enzymes - a hydroxylase (this strain also contains a chromosomal gene coding for a hydroxylase) and a dehydrogenase, located on a CAM plasmid (Chakrabarty et al., 1973). 

The branched-chain fatty acid, phytanic acid, is not a substrate for acyl CoA dehydrogenase due to the methyl group on its third (P) carbon (Figure 16.22). Instead, it is hydroxylated at the a-carbon by fatty acid a-hydroxylase. The product is decarboxylated and then activated to its CoA derivative, which is a substrate for the enzymes of P-oxidation. [Note Refsum disease is a rare, autosomal recessive disorder caused by a deficiency of a-hydroxylase. This results in the accumulation of phytanic acid in the plasma and tissues. The symptoms are primarily neurologic, and the treatment involves dietary restriction to halt disease progression.]... 

Tissue where bile acids are synthesized, and the regulated step Bile acids are synthesized in the liver. The rate-limiting step is catalyzed by cholesterol-7-a-hydroxylase, which is activated by cholesterol and inhibited by bile acids. 

In the active site of a hydroxylase, an OH group can be transferred from the peroxide to a suitable substrate (Eq. 18-42). Although radical mechanisms are likely to be involved, such hydroxylation reactions can also be viewed as transfer of OH+ to the substrate together with protonation on the inner oxygen atom of the original peroxide to give a 4a - OH adduct. The latter is a covalent hydrate which can be converted to the oxidized flavin by elimination of H20. This hydrate is believed to be the third spectral intermediate identified during the action of p-hydroxybenzoate hydroxylase 286 287 290... 

Tyrosinase is both an oxidase and a hydroxylase. Some other copper enzymes have only a hydroxylase function. One of the best understood of these is the peptidylglycine a-hydroxylating monoxygenase, which catalyzes the first step of the reaction of Eq. 10-11. The enzyme is a colorless two-copper protein but the copper atoms are 1.1 nm apart and do not form a binuclear center.570 Ascorbate is an essential cosubstrate, with two molecules being oxidized to the semidehydro-ascorbate radical as both coppers are reduced to Cu(I). A ternary complex of reduced enzyme, peptide, and 02 is formed and reacts to give the hydroxylated product.570 A related two-copper enzyme is dopamine (J-monooxygenase, which utilizes 02 and ascorbate to hydroxylate dopamine to noradrenaline (Chapter 25).571/572 These and other types of hydroxylases are compared in Chapter 18. 

Degradation of L-arginine by Streptomyces griseus is initiated by a hydroxylase that causes decarboxylation and conversion of the amino acid into an amide (Eq. 24-26), a reaction analogous to that catalyzed by the flavin-dependent lysine oxygenase (Eq. 18-41). The... 

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