Proline hydroxylases

Syntheses on the basis of 4- and 3-hydroxyprolines using regio- and stereospecific proline hydroxylases 99YGK523. 

Other examples of a-keto acid-dependent enzymes are mammalian proline hydroxylase and bacterial clavaminate synthase [113]. The latter enzyme is of particular interest as it is responsible for the catalysis of three individual steps in the biosynthesis of the (3-lactamase inhibitor clavulanic acid (Scheme 10.30). 

Hydroxylation of proline Iron proline hydroxylase (animals)... 

Procollagen proline 4-dioxygenase [Fe, ascorbate]— proline hydroxylase ... 

Vermisporin (41) is produced by the fungus Ophiobolus vermisporis [76]. Its structure was determined by chemical degradation to the derivative (42) which was studied by X-ray crystallography and provided the absolute configuration [77]. Vermisporin exhibits antimicrobial activity towards Bacteroides spp (0.25-2 lg/ml), Clostridium perfringens (0.25-2 pg/ml) and methicillin-resistant Staphylococcus aureus (0.12-0.5 pg/ml). A metabolite of Ophiobolus rubellus produces the tetramic acid (43) that has been claimed to be an inhibitor of proline hydroxylase (IC5019pM) [78]. 

Ascorbic acid (vitamin C fig. 10.16) is the reducing agent required to maintain the activity of a number of enzymes, most notably proline hydroxylase, which forms 4-hydroxyproline residues in collagen. Hydroxyproline (see fig. 10.16c) is not synthesized biologically as a free amino acid but rather is created by modification of proline residues already incorporated into collagen. The hydroxylation reaction occurs as the protein is synthesized in the endoplasmic reticulum. At least a third of the numerous proline residues in collagen are modified in this way, substantially increasing the resistance of the protein to thermal denaturation. 

Proline hydroxylase is a diooxygenase that requires ferrous iron as a cofactor and uses a-ketoglutarate as its second substrate. One oxygen atom from 02 is incorporated into hydroxyproline the other goes to the a-ketoglutarate, which decomposes to succinate and C02 ... 

Vineomycin A. (Os-4742A, P-1894B). Active against Gram-positive bacteria, collagen proline hydroxylase, and sarcoma SI80 isolated from... 

EA Popenoe, RB Aronson, DD Van Slyke. The sulfhydryl nature of collagen proline hydroxylase. Arch Biochem Biophys 133 286-292, 1969. 

RE Rhoads, S Udenfriend. Purification and properties of collagen proline hydroxylase from newborn rat skin. Arch Biochem Biophys 139 329-339, 1970. 

One-third of the amino acid residues in collagen are Gly, while another quarter are Pro. The hydroxylated amino acids 4-hydroxyproline (Hyp) and 5-hydroxylysine (Hyl) are formed post-translationally by the action of proline hydroxylase and lysine hydroxylase. These Fe2+-containing enzymes require ascorbic acid (vitamin C) for activity. In the vitamin C deficiency disease scurvy, collagen does not form correctly due to the inability to hydroxylate Pro and Lys. Hyl residues are often post-translationally modified with carbohydrate. 

Fe Fe2+, Fe3+ 1-2 mg Proline hydroxylase, diphosphoribonucleo-side dehydrogenase, peroxidases Component of hemoglobin and all other heme proteins component of iron-sulfur proteins (ferredoxins)... 

Figure 8.5 Hydroxylation of proline by proline hydroxylase to produce 4-hydroxyproline residues. (Reproduced by permission from Bezkorovainy A. Biochemistry of Nonheme Iron. New York Plenum, 1980, p. 409.)...

Proline hydroxylase (plant) mononuclear iron cell wall and lectin formation Substrate carbanion and iron bound hydroxyl 3.2.2. 

Progressive fibro-proliferative diseases (e.g. liver cirrhosis, pulmonary fibrosis, rheumatoid arthritis) result in a dramatic increase in collagen synthesis [227], This is preceded by inflammation that correlates with an increased activity of proline and lysine hydroxylase [228], Although they are unlikely to be the primary initiators of these diseases the increased activities of these enzymes may cause other problems. For example, in vitro the enzyme can turn over in the absence of a peptide substrate (but the presence of the 2-oxoglutarate cofactor). In this case stoichiometric amounts of ascorbate are required, probably to reduce the ferryl ion back to ferrous [229]. In vivo, lower concentrations of ascorbate are utilised [229,230], possibly to reactivate the enzyme after a non-productive activation (for example in the presence of a peptide that can bind to the active site, but cannot be hydroxylated). As the amount of proline-hydroxylase activity increases in the fibro-proliferative diseases, the concentration of ascorbate might not be sufficient to reduce these inactive complexes, resulting in the formation of potentially reactive ferryl intermediates. 

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). 

A. Metalloenzyme-mediated modification Proline hydroxylase Fe Y -hydroxyproline... 

Proline hydroxylase has been isolated and characterized and is known to contain a mononuclear nonheme ferrous iron center that is the catalytic active site of the enzyme, coordinated by two histidine and one aspartate side chains. The requirement for Fe(II) is reflected in the in vivo sensitivity of collagen formation to chelators specific for ferrous ion (e g. 2,2 -dipyridyl). In addition to the catalytic metal cofactor, the reaction requires a reducing cosubstrate, 2-oxoglutarate, dioxygen, and the procollagen peptide (equation 1). 

Proline hydroxylase Posttranslational Hydroxylation of proline Crosslinking of collagen... 

Fig. 7.4 Catalytic action of proline hydroxylase. One atom of oxygen gas (red) oxidizes ketoglutarate and appears in succinate along with COr The other oxygen atom forms a Fe3+-OH complex attached to the enzyme. When Fe3+ is reduced by ascorbate, H+ dissociates and the O-1 species (red) is released to a proline residue in the polypeptide, forming a hydroxyproline residue. The enzymes recognize prolyl- or lysyl residue sequence motifs described in the text (From Fig. 11.3 in Biochemistry, L. Stryer,...

Fig. 7.8 Top half of the figure depicts the spontaneous oxidation of ascorbate by oxygen-free radicals, peroxides and proline hydroxylase (black arrow) and the reduction of dehydroascorbate to ascorbate by dehydroascorbate via PDI, or providing reducing equivalents as a cofactor for peroxidases and other reductases (upper and lower red arrows). The oxidized form (GSSG) is reduced by NADPH (straight blue arrow). (Slightly modified from Meister A., Glutathione-ascorbic acid antioxidant system in animals. J. Biol. Chem., 269(13) 9397-9400,1994)...

Vitamin C (ascorbic acid, Fig. 2) is a water-soluble vitamin that dissociates at physiological pH. It is essential as a cofactor of several enzymes, including proline hydroxylase and lysine hydroxylase. Scurvy is known as the result of malnutrition with ascorbic acid. This vitamin deficiency is characterized by instable collagen. This results from insufficient hydroxylation of collagen molecules. Besides this, ascorbic acid has a function as an antioxidant. 

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