Amino cofactor-independent

Cofactor-independent Racemases and Epimerases Acting on Amino Acids... 

In a related fashion, asymmetric amination of ( )-cinnamic acid yields L-phenylalanine using L-phenylalanine ammonia lyase [EC 4,3,1,5] at a capacity of 10,000 t/year [1274, 1601], A fascinating variant of this biotransformation consists in the use of phenylalanine aminomutase from Taxus chinensis (yew tree), which interconverts ot- to p-phenylalanine in the biochemical route leading to the side chain of taxol [1602], In contrast to the majority of the cofactor-independent C-0 and C-N lyases discussed above, its activity depends on the protein-derived internal cofactor 5-methylene-3,5-dihydroimidazol-4-one (MIO) [1603], Since the reversible a,p-isomerization proceeds via ( )-cinnamic acid as achiral intermediate, the latter can be used as substrate for the amination reaction. Most remarkably, the ratio of a- vs, 3-amino acid produced (which is 1 1 for the natural substrate, R = H) strongly depends on the type and the position of substituents on the aryl moiety While o-substituents favor the formation of a-phenylalanine derivatives, / -substituted substrates predominantly lead to p-amino analogs, A gradual switch between both pathways occurred with m-substituted compounds. With few exceptions, the stereoselectivity remained exceUent (Scheme 2,215) [1604, 1605],... 

We recently succeeded in determining the three-dimensional (3D) structure of AMDase by X-ray crystallography [16]. AMDase is structurally similar to cofactor-independent amino acid racemases [17, 18]. Considering the similarities of ligand... 

Racemases are enzymes that catalyze the inversion of the chiral center by deprotonation of the C , followed by reprotonation on the opposite face of the planar carban-ionic transition-state species [13,14], In order to overcome the high energetic barrier of racemization, for example, on a-amino acids, some racemases employ pyridoxal phosphate (PLP) as a cofactor to use the resonance-stabilized amino acid complex as an electron sink because the estimated pK values for the C of amino acids are high, in the range 21-32 [14,15]. The formation of an imine PLP-substrate covalent bond makes the pK value of a-hydrogen of amino acids low. The second class of enzymes includes proline, aspartate, and glutamate racemases and diaminopimelate epimer-ase, with a cofactor-independent two-base mechanism [14],... 

A transferase that also has aldolase activity and has been used to prepare a number of chiral compounds is the enzyme serine hydroxymethyltransferase (SHMT) (EC 2.1.2.1). This enzyme, also known as threonine aldolase, catalyzes the physiological reaction of the interconversion of serine and glycine with pyridoxal phosphate (PLP) and tetrahydrofolate (FH4) as the shuttling cofactor of the C-1 unit. It also catalyzes a number of other reactions, some of which are independent of PLP and FH4 [72]. The SHMT-catalyzed aldolase reaction generates two stereocenters, which it does stereospecifically at the (/.-carbon, whereas it is less strict at the [l-carbon (Scheme 13). Nevertheless, this enzyme from porcine liver, Escherichia coU and Candida humicola (threonine aldolase) has been used to prepare a number of P-hydroxy-a-amino acids [73-76],... 

Rudnick and Abeles purified proline racemase to 95% homogeneity from Clostridium sticklandii, and characterized it 92. The enzyme is composed of two identical subunits with a molecular weight of about 38000, and is independent of any cofactors or metals. Most amino acid racemases require pyridoxal 5 -phosphate, which labilizes the bond between the a-hydrogen and the chiral center by aldimine formation with the a-amino group of the substrate. However, PLP is not involved in the reaction of proline racemase acting on an a-imino acid. The enzyme also acts on 2-hydroxy-L-proline and 2-allo-hydroxy-D-proline although slowly they are epimer-ized at a rate of 2 and 5% of the rate of L-proline racemization, respectively. L-Proline and D-proline showed Km values of 2.9 and 2.5 mti, respectively1119. ... 

A broad range of enzymic reactions of amino acids is mediated by pyridoxal phosphate (PLP), the cofactor form of vitamin Bg. The general mechanism of action of this cofactor was suggested independently by Braunstein and Shemyakin (16) and by Snell (17), and although a great deal of experimental work has been achieved since the Braunstein-Snell hypothesis was put forward, it still holds good today. 

Pyridoxal Phosphate, Codecarboxylase. An independent approach to the nature of the amino acid decarboxylases was made by Gunsalus, Umbreit, and collaborators. They found that the production of tyrosine decarboxylase by Streptococcus faecalis depended on the vitamin, pyri-doxine. In the absence of pyridoxine the cells grew but had little decarboxylase. However, addition of the vitamin permitted deficient cells to decarboxylate tyrosine, and dried cells exhibited active enzyme in the presence of pyridoxal (a derivative of pyridoxine) and ATP, implying the formation of an active cofactor from these substances. Pyridoxal is a more active growth factor for a strain of Streptococcus faecalis than pyridoxine both synthetic pyridoxal and pyridoxamine exhibit 5000 to 9000 times the activity of the hydroxy compound. ... 

A summary of the major findings on the incorporation of labeled amino acids into proteins is (1) every tissue that has been tested has been found to incorporate into its proteins, in vitro as well as in vivo, every common labeled amino acid (n-form) presented to it (2) so far one amino acid which is not a normal protein constituent—ethionine—has been found to be incorporated, two others—a-aminoadipic acid and a-aminobutyric acid —were not incorporated (3) the rate of incorporation varies with the amino acid and the tissue from approximately 0.1 to 10 micromoles per gram of protein per hour (4) the incorporation of a single amino acid appears to be largely independent of the presence of others except for specific accelerating effects of a few amino acids (5) labeled amino acids are incorporated by peptide linkage (6) in most but not all cases inhibitors of respiration and phosphorylation inhibit amino acid incorporation and protein synthesis (7) there is evidence that in some cases cofactors (unidentified) are involved. 

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