Functional expression in Escherichia coli of the tyrosine-inducible tyrosine ammonia-lyase enzyme from yeast Trichosporon cutaneum for production of p-hydroxycinnamic acid
ABSTRACT Deamination of aromatic amino acids phenylalanine or tyrosine is accomplished by the phenylalanine/tyrosine ammonia-lyase (PAL/TAL) enzyme. TAL enzyme is of interest since deamination of tyrosine produces p-hydroxycinnamic acid (pHCA), which has potential for a variety of applications. Among nine microorganisms tested for their ability to produce tyrosine-inducible TAL activity, the yeast, Trichosporon cutaneum showed the highest TAL catalytic activity and the lowest PAL/TAL catalytic efficiency ratio (0.8). The enzyme was purified to near homogeneity and its kinetics studied. The native enzyme appears to be a homo-tetramer with a calculated MW of 294 kDa, subunit MW of 73.5 kDa, and a pI of 5.8. When phenylalanine was used as substrate, the Vmax, Kcat and Km were ∼4.0 ± 0.2 μg/min/mg purified enzyme), 588 ± 29 per min and 4.9 ± 0.9 mM, respectively. However, when tyrosine served as the substrate the Vmax and Kcat were 0.59 ± 0.02 μg/min/mg purified enzyme), and 86.7 ± 29 per min, and substrate binding was apparently cooperative (nH ∼ 2.6 ± 0.4), with S0.5 ∼ 0.6 mM. This is the first reported positive cooperativity for a TAL enzyme. Based on the NH2-terminal and partial internal peptide sequences, the cDNA encoding the enzyme was cloned. Sequence analysis of TcTAL showed 56–62% similarity to other fungal PAL/TAL enzymes. High-level expression (∼30% of total soluble protein, based on SDS-PAGE analysis) of the cDNA in Escherichia coli was achieved using the arabinose inducible araB promoter. The recombinant enzyme possessed both PAL and TAL activities, as evident from the presence of both pHCA and CA in the culture medium.
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ABSTRACT: Polycyclic aromatic polyketides, such as the tetracyclines and anthracyclines, are synthesized by bacterial aromatic polyketide synthases (PKSs). Such PKSs contain a single set of iteratively used individual proteins for the construction of a highly labile poly-beta-carbonyl intermediate that is cyclized by associated enzymes to the core aromatic polyketide. A unique polyketide biosynthetic pathway recently identified in the marine strain 'Streptomyces maritimus' deviates from the normal aromatic PKS model in the generation of a diverse series of chiral, non-aromatic polyketides. A 21.3 kb gene cluster encoding the biosynthesis of the enterocin and wailupemycin family of polyketides from 'S. maritimus' has been cloned and sequenced. The biosynthesis of these structurally diverse polyketides is encoded on a 20 open reading frames gene set containing a centrally located aromatic PKS. The architecture of this novel type II gene set differs from all other aromatic PKS clusters by the absence of cyclase and aromatase encoding genes and the presence of genes encoding the biosynthesis and attachment of the unique benzoyl-CoA starter unit. In addition to the previously reported heterologous expression of the gene set, in vitro and in vivo expression studies with the cytochrome P-450 EncR and the ketoreductase EncD, respectively, support the involvement of the cloned genes in enterocin biosynthesis. The enterocin biosynthesis gene cluster represents the most versatile type II PKS system investigated to date. A large series of divergent metabolites are naturally generated from the single biochemical pathway, which has several metabolic options for creating structural diversity. The absence of cyclase and aromatase gene products and the involvement of an oxygenase-catalyzed Favorskii-like rearrangement provide insight into the observed spontaneity of this pathway. This system provides the foundation for engineering hybrid expression sets in the generation of structurally novel compounds for use in drug discovery.Chemistry & Biology 01/2001; 7(12):943-55. · 6.16 Impact Factor
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ABSTRACT: Highly purified enzyme (EC 188.8.131.52) from Rhodotorula glutinis was shown by sodium dodecyl sulfate gel electrophoresis to have subunits which if not identical are closely similar in molecular weight. Like the enzyme from maize and potato [Havir, E. A., and Hanson, K. R. (1973), Biochemistry 12, 1583] it is a tetramer of molecular weight similar to 4 times 83,000. Enzyme from all three sources inactivated and labeled at the active site with 14-CH3NO2 gave on HCl hydrolysis 14-CO2, H-14-CO2H, D- and L-[14-C]aspartic acid, and unidentified radioactive products. In addition, the labeled R. glutinis enzyme gave [1,2-14-C2]glycine. The formation of the first three products is compatible with the hypothesis that the electrophilic prosthetic group of the enzyme contains the dehydroalanine imine system greater than C equals to N minus C-alpha(equals to C-beta-H2)COminus and inactivation involves attack on C-beta. The second-order rate constants for CH3NO2 inactivation varied with pH as a simple titration curve. The pKa values calculated from the curves for the three enzymes differed and were lower than the pKa of CH3NO2 by at least 1 pH unit. Apparently the inactivation process is enzyme catalyzed. Both inactivation and addition of the substrate amino group may occur with attack on C-beta.Biochemistry 05/1975; 14(8):1620-6. · 3.38 Impact Factor
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ABSTRACT: The saccharomicins A and B, produced by the actinomycete Saccharothrix espanaensis, are oligosaccharide antibiotics. They consist of 17 monosaccharide units and the unique aglycon N-(m,p-dihydroxycinnamoyl)taurine. To investigate candidate genes responsible for the formation of trans-m,p-dihydroxycinnamic acid (caffeic acid) as part of the saccharomicin aglycon, gene expression experiments were carried out in Streptomyces fradiae XKS. It is shown that the biosynthetic pathway for trans-caffeic acid proceeds from L-tyrosine via trans-p-coumaric acid directly to trans-caffeic acid, since heterologous expression of sam8, encoding a tyrosine ammonia-lyase, led to the production of trans-p-hydroxycinnamic acid (coumaric acid), and coexpression of sam8 and sam5, the latter encoding a 4-coumarate 3-hydroxylase, led to the production of trans-m,p-dihydroxycinnamic acid. This is not in accordance with the general phenylpropanoid pathway in plants, where trans-p-coumaric acid is first activated before the 3-hydroxylation of its ring takes place.Journal of Bacteriology 05/2006; 188(7):2666-73. · 3.19 Impact Factor