Bacterial formate dehydrogenase. Increasing the enzyme thermal stability by hydrophobization of alpha-helices.

NAD+-dependent formate dehydrogenase (EC 1.2.1.2, FDH) from methylotrophic bacterium Pseudomonas sp.101 exhibits the highest stability among the similar type enzymes studied. To obtain further increase in the thermal stability of FDH we used one of general approaches based on hydrophobization of protein alpha-helices. Five serine residues in positions 131, 160, 168, 184 and 228 were selected for mutagenesis on the basis of (i) comparative studies of nine FDH amino acid sequences from different sources and (ii) with the analysis of the ternary structure of the enzyme from Pseudomonas sp.101. Residues Ser-131 and Ser-160 were replaced by Ala, Val and Leu. Residues Ser-168, Ser-184 and Ser-228 were changed into Ala. Only Ser/Ala mutations in positions 131, 160, 184 and 228 resulted in an increase of the FDH stability. Mutant S168A was 1.7 times less stable than the wild-type FDH. Double mutants S(131,160)A and S(184,228)A and the four-point mutant S(131,160,184,228)A were also prepared and studied. All FDH mutants with a positive stabilization effect had the same kinetic parameters as wild-type enzyme. Depending on the position of the replaced residue, the single point mutation Ser/Ala increased the FDH stability by 5-24%. Combination of mutations shows near additive effect of each mutation to the total FDH stabilization. Four-point mutant S(131,160,184,228)A FDH had 1.5 times higher thermal stability compared to the wild-type enzyme.

Site-directed mutagenesis of the formate dehydrogenase active centre: role of the His332-Gln313 pair in enzyme catalysis.

Gln313 and His332 residues in the active centre of NAD(+)-dependent formate dehydrogenase (EC 1.2.1.2, FDH) from the bacterium Pseudomonas sp. 101 are conserved in all FDHs and are equivalent to the glutamate-histidine pair in active sites of D-specific 2-hydroxyacid dehydrogenases. Two mutants of formate dehydrogenase from Pseudomonas sp. 101, Gln313Glu and His332Phe, have been obtained and characterised. The Gln313Glu mutation shifts the pK of the group controlling formate binding from less than 5.5 in wild-type enzyme to 7.6 thus indicating that Gln313 is essential for the broad pH affinity profile towards substrate. His332Phe mutation leads to a complete loss of enzyme activity. The His332Phe mutant is still able to bind coenzyme but not substrate or analogues. The role of histidine in the active centre of FDH is discussed. The protonation state of His332 is not critical for catalysis but vital for substrate binding. A partial positive charge on the histidine imidazole, required for substrate binding, is provided via tight H-bond to the Gln313 carboxamide.

Evidence for indole-3-acetic acid binding site in plant peroxidases. Structural similarity between peroxidases and auxin-binding proteins.

Application of computer methods allowed us to demonstrate that plant peroxidases and auxin-binding proteins contain structurally similar fragments. The mapping of the fragments was done using a model structure of horseradish peroxidase. Five of six structurally similar fragments belong to the distal domain and form a subdomain in plant peroxidases that includes the distal heme-coordinating sequence, LHFHDC (amino acid residues 39-44 in horseradish peroxidase). The existence of a substrate-binding site for indole-3-acetic acid in the distal subdomain comprising helices A (whole), B (middle), C (beginning), and D (whole) and the loop between helices D and D' is discussed.

Oxidation of indole-3-acetic acid by dioxygen catalysed by plant peroxidases: specificity for the enzyme structure.

Indole-3-acetic acid (IAA) can be oxidized via two mechanisms: a conventional hydrogen-peroxide-dependent pathway, and one that is hydrogen-peroxide-independent and requires oxygen. It has been shown here for the first time that only plant peroxidases are able to catalyse the reaction of IAA oxidation with molecular oxygen. Cytochrome c peroxidase (CcP), fungal peroxidases (manganese-dependent peroxidase, lignin peroxidase and Arthromyces ramosus peroxidase) and microperoxidase were essentially inactive towards IAA in the absence of added H2O2. An analysis of amino acid sequences allowed five structurally similar fragments to be identified in auxin-binding proteins and plant peroxidases. The corresponding fragments in CcP and fungal peroxidases showed no similarity with auxin-binding proteins. Five structurally similar fragments form a subdomain including the catalytic centre and two residues highly conserved among 'classical' plant peroxidases only, namely His-40 and Trp-117. The subdomain identified above with the two residues might be responsible for the oxidation of the physiological substrate of classical plant peroxidases, IAA.

Alternative cyclization in GFP-like proteins family. The formation and structure of the chromophore of a purple chromoprotein from Anemonia sulcata.

Anemonia sulcata purple protein (asFP595) belongs to a family of green fluorescent protein (GFP)-like proteins from the Anthozoa species. Similar to GFP, asFP595 apparently forms its chromophore by modifying amino acids within its polypeptide chain. Until now, the GFP-like proteins from Anthozoa were thought to contain chromophores with the same imidazolidinone core as GFP. Mass spectral analysis of a chromophore-containing tryptic pentapeptide from asFP595 demonstrates that chromophore formation in asFP595 is stoichiometrically the same as that in GFP: one H(2)O and two H(+) are released while a Schiff base and dehydrotyrosine are formed. However, structural studies of this asFP595 chromopeptide show that in contrast to GFP, the other peptide bond nitrogen and carbonyl carbon are required for chromophore cyclization, a reaction that yields the six-membered heterocycle 2-(4-hydroxybenzylidene)-6-hydroxy-2,5-dihydropyrazine. Spectrophotometric titration reveals three pH-dependent forms of the asFP595 chromopeptide: yellow (absorption maximum = 430 nm) at pH 3.0; red (absorption maximum = 535 nm) at pH 8.0; and colorless (absorption maximum = 380 nm) at pH 14.0. The pK(a) values for these spectral transitions (6.8 and 10.9) are consistent with the ionization of the phenolic group of dehydrotyrosine and deprotonation of the amidinium cation in the chromophore heterocycle, respectively. The amidinium group in asFP595 accounts for the unique absorption spectrum of the protein, which is substantially red-shifted relative to that of GFP. When the asFP595 chromophore cyclizes, the Cys-Met bond adjacent to the chromophore hydrolyzes, splitting the chromoprotein into 8- and 20-kDa fragments. High performance liquid chromatography analysis of a tryptic digest of denatured asFP595 shows that a pentapeptide with the cleaved Cys-Met bond is the only fragment associated with the red-shifted absorbance. These results imply that fragmentation of asFP595 is a critical step in protein maturation.

Catalytic properties of tryptophanless recombinant horseradish peroxidase.

Heme-containing plant peroxidases (EC 1.11.1.7) contain a highly conserved single tryptophan residue. Its replacement with Phe in recombinant horseradish peroxidase (rHRP) increased the stability of the mutant enzyme in acid media. The kinetic properties of native, wild-type, and W117F mutant recombinant horseradish peroxidase in the reactions of ammonium 2, 2;-azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS), guaiacol, and o-phenylenediamine oxidation are very similar. However, significant changes in the reaction rate constant characteristic for the monomolecular rate-limiting step ascribed either to product dissociation from its complex with the enzyme or electron transfer from the substrate to the active site within the Michaelis complex were observed. The data indirectly indicate the participation of the single Trp residue in oxidation of ABTS and guaiacol and possible differences in kinetic mechanisms for oxidation of ABTS, guaiacol, and o-phenylenediamine.

Pilot scale production and isolation of recombinant NAD+- and NADP+-specific formate dehydrogenases.

The expression of the recombinant wild-type NAD+- and mutant NADP+-dependent formate dehydrogenases (EC 1.2.1.2., FDH) from the methanol-utilizing bacterium Pseudomonas sp. 101 in Escherichia coli cells has been improved to produce active and soluble enzyme up to the level of 50% of total soluble proteins. The cultivation process for E. coli/pFDH8a and E. coli/pFDH8aNP cells was optimized and scaled up to a volume of 100 L. A downstream purification process has been developed to produce technical grade NAD+- and NADP+-specific formate dehydrogenases in pilot scale, utilizing extraction in aqueous two-phase systems.

Tryptophanless recombinant horseradish peroxidase: stability and catalytic properties.

The tryptophanless mutant of horseradish peroxidase, W117F, has been constructed and expressed in Escherichia coli. The mutation affects enzyme folding and stability. The optimum composition of the refolding medium requires the presence of ammonium sulfate. The yield of mutant is ca. 8000 U per liter of the optimized refolding medium with the specific activity of 1100-1500 U/mg (compared to 25, 000 U per liter and 2000 U/mg for the recombinant wild-type enzyme). The mutant is more stable in acid media, in the reaction course and toward irradiation. The effect of hydrogen peroxide pretreatment on radiation-induced inactivation of the wild-type and mutant enzyme indirectly indicates participation of Trp-117 in electron transfer pathways through the enzyme molecule. This is in agreement with the steady-state kinetic data interpreted in terms of Trp-117 participation in electron transfer within the Michaelis complex.