Collagen is the main structural protein in vertebrates. It plays an essential role in providing a scaffold for cellular support and thereby affecting cell attachment, migration, proliferation, differentiation, and survival. As such, it also plays an important role in numerous approaches to the engineering of human tissues for medical applications related to tissue, bone, and skin repair and reconstruction. Currently, the collagen used in tissue engineering applications is derived from animal tissues, creating concerns related to the quality, purity, and predictability of its performance. It also carries the risk of transmission of infectious agents and precipitating immunological reactions. The recent development of recombinant sources of human collagen provides a reliable, predictable and chemically defined source of purified human collagens that is free of animal components. The triple-helical collagens made by recombinant technology have the same amino acid sequence as human tissue-derived collagen. Furthermore, by achieving the equivalent extent of proline hydroxylation via coexpression of genes encoding prolyl hydroxylase with the collagen genes, one can produce collagens with a similar degree of stability as naturally occurring material. The recombinant production process of collagen involves the generation of single triple-helical molecules that are then used to construct more complex three-dimensional structures. If one loosely defines tissue engineering as the use of a biocompatible scaffold combined with a biologically active agent (be it a gene or gene construct, growth factor or other biologically active agent) to induce tissue regeneration, then the production of recombinant human collagen enables the engineering of human tissue based on a human matrix or scaffold. Recombinant human collagens are an efficient scaffold for bone repair when combined with a recombinant bone morphogenetic protein in a porous, sponge-like format, and when presented as a membrane, sponge or gel can serve as a basis for the engineering of skin, cartilage and periodontal ligament, depending on the specific requirements of the chosen application.

Tyrosine and phenylalanine hydroxylases contain homologous catalytic domains and dissimilar regulatory domains. To determine the effects of the regulatory domains upon the substrate specificities, truncated and chimeric mutants of tyrosine and phenylalanine hydroxylase were constructed: Delta117PAH, the C-terminal 336 amino acid residues of phenylalanine hydroxylase; Delta155TYH, the C-terminal 343 amino acid residues of tyrosine hydroxylase; and 2 chimeric proteins, 1 containing the C-terminal 331 residues of phenylalanine hydroxylase and the N-terminal 168 residues of tyrosine hydroxylase, and a second containing the C-terminal 330 residues of tyrosine hydroxylase and the 122 N-terminal residues of phenylalanine hydroxylase. Steady-state kinetic parameters with tyrosine and phenylalanine as substrate and the need for pretreatment with phenylalanine for full activity were determined. The truncated proteins showed low binding specificity for either amino acid. Attachment of either regulatory domain greatly increased the specificity, but the specificity was determined by the catalytic domain in the chimeric proteins. All three proteins containing the catalytic domain of phenylalanine hydroxylase were unable to hydroxylate tyrosine. Only wild-type phenylalanine hydroxylase required pretreatment with phenylalanine for full activity with tetrahydrobiopterin as substrate.

The peroxiredoxin AhpC from Mycobacterium tuberculosis has been expressed, purified, and characterized. It differs from other well characterized AhpC proteins in that it has three rather than one or two cysteine residues. Mutagenesis studies show that all three cysteine residues are important for catalytic activity. Analysis of the M. tuberculosis genome identified a second protein, AhpD, which has no sequence identity with AhpC but is under the control of the same promoter. This protein has also been cloned, expressed, purified, and characterized. AhpD, which has only been identified in the genomes of mycobacteria and Streptomyces viridosporus, is shown here to also be an alkylhydroperoxidase. The endogenous electron donor for catalytic turnover of the two proteins is not known, but both can be turned over with AhpF from Salmonella typhimurium or, particularly in the case of AhpC, with dithiothreitol. AhpC and AhpD reduce alkylhydroperoxides more effectively than H 2 O 2 but do not appear to interact with each other. These two proteins appear to be critical elements of the antioxidant defense system of M. tuberculosis and may be suitable targets for the development of novel anti-tuberculosis strategies.

Tyrosine hydroxylase is an iron-containing monooxygenase that uses a tetrahydropterin to catalyze the hydroxylation of tyrosine to dihydroxyphenylalanine in catecholamine biosynthesis. The role of the iron in this enzyme is not understood. Purification of recombinant rat tyrosine hydroxylase containing 0.5-0.7 iron atoms/ subunit and lacking bound catecholamine has permitted studies of the redox states of the resting enzyme and the enzyme during catalysis. As isolated, the iron is in the ferric form. Dithionite or 6-methyltetrahydropterin can reduce the iron to the ferrous form. Reduction by 6-methyltetrahydropterin consumes 0.5 nmol/nmol of enzyme-bound iron, producing quinonoid 6-methyldihydropterin as the only detectable product. In the presence of oxygen, reoxidation to ferric iron occurs. During turnover the enzyme is in the ferrous form. However, a fraction is oxidized during turnover; this can be trapped by added catechol or by the dihydroxyphenylalanine formed during turnover.Tyrosine hydroxylase catalyzes the hydroxylation of tyrosine to form dihydroxyphenylalanine (DOPA), 1 the rate-limiting step in the biosynthesis of the catecholamine neurotransmitters (1). This is one of a small family of tetrahydropterinutilizing monooxygenases found in the central nervous system; the others are phenylalanine hydroxylase and tryptophan hydroxylase. The mechanisms of these enzymes are very poorly understood, and there are as yet no structures available. Tyrosine hydroxylase has been known for some time to contain 1 iron atom/subunit (2). Studies of the metal dependence of the catalytic activity have shown that ferrous iron is required; no other metal has been found to be catalytically active (3, 4). The iron atom is bound to amino acid side chains rather than a porphyrin ring; recently two of the metal ligands in rat tyrosine hydroxylase have been identified as histidines 331 and 336 (5). As yet, the role of the iron in tyrosine hydroxylase remains unclear. However, the lack of activity of iron-depleted enzyme (3, 4), or of enzyme in which a metal ligand has been modified by site-directed mutagenesis (5), is consistent with an essential role in catalysis. In addition, NMR measurements have shown that the amino acid substrate binds close to the iron, placing the metal in the active site (6).Until relatively recently, studies of the metal site in tyrosine hydroxylase were hindered by the difficulties of obtaining sufficient amounts of purified enzyme for study. Within the last decade preparations from bovine adrenal medulla (7) and rat pheochromocytoma (8) have permitted physical studies. The enzyme from both sources contained 0.6 -0.7 iron atoms/subunit. EPR spectroscopy showed that the iron was in the iron-(III) state when isolated. Furthermore, the enzyme had a bluegreen color due to the presence of tightly bound catecholamines interacting with the metal (8, 9). More recently, several laboratories have successfully expressed human or rat tyrosine hydroxylase in bacteria, providing access to significantly more mater...

The iron-containing enzyme tyrosine hydroxylase catalyzes the hydroxylation of tyrosine to dihydroxyphenylalanine. A series of 4-X-substituted (X = H, F, Br, Cl, CH3, or CH3O) phenylalanines have been characterized as substrates to gain insight into the mechanism of hydroxylation. Multiple hydroxylated products were formed in most cases. As the size of the substituent at the 4-position increased, the site of hydroxylation switched from the 4- to the 3-position of the aromatic ring. The total amount of product formed with each amino acid showed a very good correlation with the sigma parameter of the substituent, with rho values of -4.3 +/- 0.7 or -5.6 +/- 0.8 when tetrahydrobiopterin or 6-methyltetrahydropterin, respectively, was used as cosubstrate. These values are consistent with a highly electron deficient transition state for hydroxylation. Oxygen addition at the 4-position resulted in either elimination of the substituent to form tyrosine or an NIH shift to form the respective 3-X-tyrosine. The relative amount of the product due to an NIH shift decreased in the order Br > CH3 > Cl >> F approximately CH3O approximately 0. A chemical mechanism for hydroxylation by tyrosine hydroxylase is presented to account for product formation from the various 4-substituted phenylalanines.