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1.
Biochim Biophys Acta ; 1804(4): 752-4, 2010 Apr.
Article in English | MEDLINE | ID: mdl-19948253

ABSTRACT

The (13)C isotope effect for the conversion of prephenate to phenylpyruvate by the enzyme prephenate dehydratase from Methanocaldococcus jannaschii is 1.0334+/-0.0006. The size of this isotope effect suggests that the reaction is concerted. From the X-ray structure of a related enzyme, it appears that the only residue capable of acting as the general acid needed for removal of the hydroxyl group is threonine-172, which is contained in a conserved TRF motif. The more favorable entropy of activation for the enzyme-catalyzed process (25 eu larger than for the acid-catalyzed reaction) has been explained by a preorganized microenvironment that obviates the need for extensive solvent reorganization. This is consistent with forced planarity of the ring and side chain, which would place the leaving carboxyl and hydroxyl out of plane. Such distortion of the substrate may be a major contributor to catalysis.


Subject(s)
Archaeal Proteins/chemistry , Archaeal Proteins/metabolism , Methanococcales/enzymology , Prephenate Dehydratase/chemistry , Prephenate Dehydratase/metabolism , Archaeal Proteins/genetics , Carbon Isotopes , Catalysis , Catalytic Domain , Entropy , Enzyme Activation , Kinetics , Methanococcales/genetics , Prephenate Dehydratase/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Threonine/chemistry
2.
Appl Microbiol Biotechnol ; 81(2): 263-73, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18704396

ABSTRACT

The alcohol dehydrogenase from Thermus sp. ATN1 (TADH) was characterized biochemically with respect to its potential as a biocatalyst for organic synthesis. TADH is a NAD(H)-dependent enzyme and shows a very broad substrate spectrum producing exclusively the (S)-enantiomer in high enantiomeric excess (>99%) during asymmetric reduction of ketones. TADH is active in the presence of 10% (v/v) water-miscible solvents like 2-propanol or acetone, which permits the use of these solvents as sacrificial substrates in substrate-coupled cofactor regeneration approaches. Furthermore, the presence of a second phase of a water-insoluble solvent like hexane or octane had only minor effects on the enzyme, which retained 80% of its activity, allowing the use of these solvents in aqueous/organic mixtures to increase the availability of low-water soluble substrates. A further activity of TADH, the production of carboxylic acids by dismutation of aldehydes, was investigated. This reaction usually proceeds without net change of the NAD(+)/NADH concentration, leading to equimolar amounts of alcohol and carboxylic acid. When applying cofactor regeneration at high pH, however, the ratio of acid to alcohol could be changed, and full conversion to the carboxylic acid was achieved.


Subject(s)
Alcohol Dehydrogenase/genetics , Alcohol Dehydrogenase/metabolism , Thermus/enzymology , 2-Propanol/pharmacology , Acetone/pharmacology , Alcohols/metabolism , Aldehydes/metabolism , Carboxylic Acids/metabolism , Coenzymes/pharmacology , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Enzyme Inhibitors/pharmacology , Hexanes/pharmacology , Ketones/metabolism , Molecular Sequence Data , NAD/pharmacology , Octanes/pharmacology , Sequence Analysis, DNA , Stereoisomerism , Substrate Specificity , Thermus/genetics
3.
Proc Natl Acad Sci U S A ; 104(35): 13907-12, 2007 Aug 28.
Article in English | MEDLINE | ID: mdl-17715291

ABSTRACT

The biosynthesis of small molecules can be fine-tuned by (re)engineering metabolic flux within cells. We have adapted this approach to optimize an in vivo selection system for the conversion of prephenate to phenylpyruvate, a key step in the production of the essential aromatic amino acid phenylalanine. Careful control of prephenate concentration in a bacterial host lacking prephenate dehydratase, achieved through provision of a regulable enzyme that diverts it down a parallel biosynthetic pathway, provides the means to systematically increase selection pressure on replacements of the missing catalyst. Successful differentiation of dehydratases whose activities vary over a >50,000-fold range and the isolation of mechanistically informative prephenate dehydratase variants from large protein libraries illustrate the potential of the engineered selection strain for characterizing and evolving enzymes. Our approach complements other common methods for adjusting selection pressure and should be generally applicable to any selection system that is based on the conversion of an endogenous metabolite.


Subject(s)
Prephenate Dehydratase/genetics , Selection, Genetic , Amino Acids/metabolism , Directed Molecular Evolution , Genetic Engineering/methods , Genetic Variation , Kinetics , Models, Genetic , Models, Molecular , Plasmids , Prephenate Dehydratase/chemistry , Prephenate Dehydratase/metabolism , Protein Conformation , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Shikimic Acid/metabolism
4.
Biochemistry ; 45(47): 14101-10, 2006 Nov 28.
Article in English | MEDLINE | ID: mdl-17115705

ABSTRACT

Prephenate dehydratase (PDT) is an important but poorly characterized enzyme that is involved in the production of L-phenylalanine. Multiple-sequence alignments and a phylogenetic tree suggest that the PDT family has a common structural fold. On the basis of its sequence, the PDT from the extreme thermophile Methanocaldococcus jannaschii (MjPDT) was chosen as a promising representative of this family for pursuing structural and functional studies. The corresponding pheA gene was cloned and expressed in Escherichia coli. It encodes a monofunctional and thermostable enzyme with an N-terminal catalytic domain and a C-terminal regulatory ACT domain. Biophysical characterization suggests a dimeric (62 kDa) protein with mixed alpha/beta secondary structure elements. MjPDT unfolds in a two-state manner (Tm = 94 degrees C), and its free energy of unfolding [DeltaGU(H2O)] is 32.0 kcal/mol. The purified enzyme catalyzes the conversion of prephenate to phenylpyruvate according to Michaelis-Menten kinetics (kcat = 12.3 s-1 and Km = 22 microM at 30 degrees C), and its activity is pH-independent over the range of pH 5-10. It is feedback-inhibited by L-phenylalanine (Ki = 0.5 microM), but not by L-tyrosine or L-tryptophan. Comparison of its activation parameters (DeltaH(++)= 15 kcal/mol and DeltaS(++)= -3 cal mol-1 K-1) with those for the spontaneous reaction (DeltaH(++) = 17 kcal/mol and DeltaS(++)= -28 cal mol-1 K-1) suggests that MjPDT functions largely as an entropy trap. By providing a highly preorganized microenvironment for the dehydration-decarboxylation sequence, the enzyme may avoid the extensive solvent reorganization that accompanies formation of the carbocationic intermediate in the uncatalyzed reaction.


Subject(s)
Methanococcus/enzymology , Prephenate Dehydratase/metabolism , Amino Acid Sequence , Base Sequence , Catalysis , Cloning, Molecular , DNA Primers , Enzyme Stability , Kinetics , Molecular Sequence Data , Prephenate Dehydratase/chemistry , Prephenate Dehydratase/genetics , Protein Folding , Protein Structure, Secondary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Thermodynamics
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