Trimethyllysine 3-hydroxylase

Figure 14.2. Biosynthesis of carnitine. Trimethyllysine hydroxylase, EC 1.14.11.8 aldolase, EC 4.1.2. x aldehyde dehydrogenase, EC 1.2.1.47 -butyrohetaine hydroxylase, EC 1.14.11.1. Relative molecnlar mass (Mr) carnitine, 161.2.

Of all the enzymes of the carnitine biosynthetic pathway, yBBH is the best-studied enzyme. Like e-N-trimethyllysine hydroxylase, y-BBH is a non-heme ferrous-iron dioxygenase that requires a-ketoglutarate, Fe and molecular oxygen as cofactors. In this class of enzymes, the hydroxylation of the substrate is linked to the oxidative decarboxylation of a-ketoglutarate. Ascorbate is needed to maintain iron in die reduced state. yBBHhas been isolated from various sources including human kidney, " catf and rat liver and the bacterium Pseudomonas AKl. A common problem in the purification of this enzyme from mammalian tissues is the poor stability of the protein. We, therefore, determined the optimal storage conditions and subsequently pmified ybutyrobetaine hydroxylase from rat liver. 

All the enzymes contain ferrous iron. Ferrous ions are essential for the catalytic activity of butyrobetaine hydroxylase (259), proline-4- 254) and 3- 242, 243), hydroxylases, thymidine hydroxylase 260), lysine hydroxylase 261), trimethyllysine hydroxylase 247), thymine oxygenase 262—264), cephalosporin hydroxylase 249), and hydroxy-phenylpyruvate 265—267). In addition, the enzymes show a non-stoichio-metric requirement for ascorbic acid which is supposed to maintain the ion co-factor in the ferrous oxidation state (2, 242, 243, 254). Labelling studies with butyrobetaine hydroxylase 268), proline-4-hydroxylase 269), thymine oxygenase (270), cephalosporin hydroxylase (257) and hydroxy-phenylpyruvate hydroxylase (277) all indicate a common pattern of oxygen incorporation. One atom of oxygen becomes the new hydroxyl group while the other is located in one of the carboxylic acid groups of succinic acid. 

Henderson, L. L., and L. M. Henderson Purification and Properties Trimethyllysine Hydroxylase. Fed. Amer. Soc. Exp. Biol. 63id Annual Meet. 1979, 2032. 

In humans, camitine is either obtained from the diet or synthesised de novo (Fig. 1). Camitine biosynthesis in higher eukaryotes starts when protein-bound L-lysine is trimethylated by a protein-dependent methyltransferase to form e-N-trimethyllysine. Upon degradation of these proteins, free e-N-trimethyllysine becomes available and is hydroxylated at the 3-position by e-N-trimethyllysine hydroxylase. Subsequently, P-hydroxy- e-N-trimethyllysine is cleaved into ytrimethylaminobutyraldehyde and glycine by p-hydroxy-e-N-trimethyllysine aldolase, after which the aldehyde is oxidized by y-trimethylaminobutyraldehyde dehydrogenase to yield y-butyrobetaine. Finally, y-buty-robetaine is hydroxylated at the 3-position by "j utyrobetaine hydroxylase (y BBH) to produce L-camitine (see Fig. 1). 

Enzymatic hydroxylation of biological molecules is often catalyzed by hydroxylases. These types of enzymes are either oxygenases or peroxidases, in which the source of oxygen is O2 or H2O2, respectively. Cytochrome P-450-dependent enzymes represent a common class of enzymes that carry out hydroxylation reactions. L-Carnitine is a metabolite isolated from many organisms and its biosynthesis begins with the enzymatic hydroxylation of trimethyllysine. The intermediate, 3-hydroxyl-e-(A(A(ALtrimethyl)-L-lysine, is further... 

Lysine is not only a constituent of proteins. It can also be trimethylated and converted to carnitine (p. 944). In mammals some specific lysyl side chains of proteins undergo N-trimethylation and proteolytic degradation with release of free trimethyllysine (Eq. 24-30) 278/279 The free trimethyllysine then undergoes hydroxylation by a 2-oxoglutarate-Fe2+-ascorbate-dependent hydroxylase (Eq. 18-51) to form P-hydroxytrimethyllysine, which is cleaved by a PLP-dependent enzyme (Chapter 14). The resulting aldehyde is oxidized to the carboxylic acid and is converted by a second 2-oxoglutarate-Fe2+-ascorbate-dependent hydroxylase to carnitine (Eq. 24-30 see also Eq. 18-50). 

As shown in Table 13.1, a number of iron-containing hydroxylases share an unusual reaction mechanism in which hydroxylation of the substrate is linked to decarboxylation of 2-oxoglutarate. Proline and lysine hydroxylases are required for the postsynthetic modification of collagen, and proline hydroxylase also for the postsynthetic modification of osteocalcin (Section 5.3.3) and other proteins. Aspartate /3-hydroxylase is required for the postsynthetic modification of protein C, the vitamin K-dependent protease that hydrolyzes activated Factor V in the blood clotting cascade (Section 5.3.2). Trimethyllysine and y-butyrobetaine hydroxylases are required for the synthesis of carnitine (Section 14.1.1). 

In general, the effects on collagen synthesis are more marked and more important than those of decreased formation of carnitine (as a result of impaired activity of trimethyllysine and y-butyrobetaine hydroxylases Section 14.1.1), impaired xenobiotic metabolism, or hypercholesterolemia (Section 13.3.8). However, depletion of muscle carititine may account for the lassitude and fatigue that precede clinical signs of scurvy. 

In general, the effects on collagen synthesis are more marked and more important than those of decreased formation of carnitine (as a result of impaired activity of trimethyllysine and y-butyrobetaine hydroxylases Section... 

Carnitine biosynthesis in humans. A lysyl residue is trimethylated by S-adenosyimethionine, with subsequent proteolytic release of trimethyllysine, the starting material. The reactions are catalyzed by (1) trimethyllysine A-hydroxylase, (2) /t-hydroxy-trimethyllysine aldolase (pyridoxal phosphate), (3) )/-trimethylaminobutyraldehyde dehydrogenase, and (4) /-butyrobetaine hydroxylase. 

Trimethyllysine and y-butyrobetaine hydroxylases are required for the synthesis of carnitine (section 5.5.1). 

T ymidine-2 -hy droxylase Trimethyllysine 3-hydroxylase Cephalosporin hydroxylase... 

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