Loredano Pollegioni

Specificity and kinetics of Rhodotorula gracillisd-amino acid oxidase

D-Amino acid oxidase purified from the yeast Rhodotorula gracilis is a flavoenzyme which does not require exogenous FAD for maximum activity. The enzyme showed temperature and pH activity optima centred between 40 and 45 degrees C and between 8.0 and 8.5, respectively; a broad pH and ionic strength range of stability and a more limited range of thermostability was determined. The enzyme stability was markedly influenced by the presence of 2-mercaptoethanol. Apparent kinetic parameters for a number of substrates were determined: nonpolar and aromatic D-amino acids appeared to be the best substrates. Steady state measurements carried out at different oxygen concentrations indicated that for D-alanine the kinetic pattern is consistent with a Ping Pong Bi Bi mechanism; kcat values on D-alanine and D-valine are 43,250 min-1 and 31,370 min-1, respectively. L-Amino acids did not inhibit enzyme activity; several aromatic and aliphatic carboxylic acids proved to be competitive inhibitors of the enzyme and their ki values were determined. The reported properties of R. gracilis D-amino acid oxidase markedly distinguish it from other characterized D-amino acid oxidases.

The primary structure of D-amino acid oxidase from Rhodotorula gracilis

SummaryThe amino acid sequence of D-amino acid oxidase from Rhodotorula gracilis was determined by automated Edman degradation of peptides generated by enzymatic and chemical cleavage. The enzyme monomer contains 368 amino acid residues and its sequence is homologous to that of other known D-amino acid oxidases. Six highly conserved regions appear to have a specific role in binding of coenzyme FAD, in active site topology and in peroxisomal targeting. Moreover, Rhodotorula gracilis D-amino acid oxidase contains a region with a cluster of basic amino acids, probably exposed to solvent, which is absent in other D-amino acid oxidases.

On the mechanism of D-amino acid oxidase : structure/linear free energy correlations and deuterium kinetic isotope effects using substituted phenylglycines

The kinetic mechanism of the reaction of D-amino acid oxidase (EC 1.4.3.3) from Trigonopsis variabilis with [α-1H]- and [α-2H]phenylglycine has been determined. The pH dependence of Vmax is compatible with pKa values of ≈8.1 and >9.5, the former of which is attributed to a base which should be deprotonated for efficient catalysis. The deuterium isotope effect on turnover is ≈3.9, and the solvent isotope effect ≈1.6. The reductive half-reaction is biphasic, the first, fast phase, k2, corresponding to substrate dehydrogenation/enzyme flavin reduction and the second to conversion/release of product. Enzyme flavin reduction consists in an approach to equilibrium involving a finite rate for k−2, the reversal of k2. k2 is 28.8 and 4.6 s−1 for [α-1H]- and [α-2H]phenylglycine, respectively, yielding a primary deuterium isotope effect ≈6. The solvent deuterium isotope effect on the apparent rate of reduction for [α-1H]- and [α-2H]phenylglycine is ≈2.8 and ≈5. The rates for k−2 are 4.2 and 0.9 s−1 for [α-1H]- and [α-2H]phenylglycine, respectively, and the corresponding isotope effect is ≈4.7. The isotope effect on α-H and the solvent one thus behave multiplicatively consistent with a highly concerted process and a symmetric transition state. The k2 and k−2 values for phenylglycines carrying the para substituents F, Cl, Br, CH3, OH, NO2 and OCH3 have been determined. There is a linear correlation of k2 with the substituent volume VM and with σ+; k−2 correlates best with σ or σ+ while steric parameters have little influence. This is consistent with the transition state being structurally similar to the product. The Brønsted plot of ΔG‡ versus ΔG0 allows the estimation of the intrinsic ΔG0‡ as ≈58 kJ·;M−1. From the linear free energy correlations, the relation of ΔG‡ versus ΔG0 and according to the theory of Marcus it is concluded that there is little if any development of charge in the transition state. This, together with the recently solved three-dimensional structure of D-amino acid oxidase from pig kidney (Mattevi, A., Vanoni, M.A., Todone, F., Rizzi, M., Teplyakov, A., Coda, A., Bolognesi, M., and Curti, B. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 7496-7501), argues against a carbanion mechanism in its classical formulation. Our data are compatible with transfer of a hydride from the substrate αC-H to the oxidized flavin N(5) position, although, clearly, they cannot prove it.

A process for bioconversion of cephalosporin C by Rhodotorula gracilis D-amino acid oxidase

SummaryA process for the production (in a stirred tank reactor) of glutaryl-7-ACA from cephalosporin C using immobilized D-amino acid oxidase is described. Results so obtained under optimal conditions (1.2 mg coupled enzyme/L, pH 8.5, 2 mM cephalosporin C) point to a system which shows high conversion efficiency and a remarkable operational stability. No exogenous H2O2 is requested to shift the reaction equilibrium toward glutaryl-7-ACA production, nor any side product is detected. The immobilized system productivity was 54 g/day/mg of enzyme. This process represents the first reported case of a reactor successfully developed with a DAAO for bioconversion of cephalosporin C.

Cloning, sequencing and expression in E. coli of a D-amino acid oxidase cDNA from Rhodotorula gracilis active on cephalosporin C.

We have cloned the cDNA coding for the Rhodotorula gracilis D-amino acid oxidase (DAAO), an enzyme that performs with high catalytic efficiency biotechnologically relevant bioconversions, by PCR amplification. The first strand cDNA was synthesised from the total mRNA fraction isolated from R. gracilis cells grown under DAAO-inducing conditions. The R. gracilis DAAO cDNA consists of 1104 bp encoding a protein of 368 amino acids. The insertion of the cDNA into the pKK223-3 plasmid allowed the expression of recombinant DAAO in Escherichia coli as a wholly soluble and catalytically active holoenzyme (approximately 0.5 U mg-1 protein) with a fermentation yield, in terms of DAAO units, of 800 U l-1. This level of expression allowed the purification, in homogeneous form and high yield (50%), of the recombinant enzyme which showed a high catalytic activity on cephalosporin C as substrate. The nucleotide sequence reported in this paper will appear in the nucleotide sequence databases under accession number.

Toxic cyanobacterial blooms in Lake Varese (Italy): A multidisciplinary approach

During the summer of 1997, a toxic cyanobacterial bloom was observed in Lake Varese. The cyanobacterial community is recognized morphologically as being dominated by species belonging to the genera Aphanizomenon, Oscillatoria, Anabaena, and Microcystis. HPLC analyses were performed on phytoplankton samples collected on the lake from July to September 1997. A HPLC peak that had the same retention time as saxitoxin was detected in the sample collected on August 19. In addition, extracts from shellfish and fish tissues showed the same peak. In electrophysiological experiments, extracts of cyanobacteria showed an almost complete inhibition of Na+ channels, the typical biological effect of saxitoxin. The genomic region corresponding to the phycocyanin intergenic spacer of cyanobacteria from the last summer bloom has been characterized by means of a PCR amplification technique. ©1999 John Wiley & Sons, Inc. Environ Toxicol 14: 127–134, 1999

IMMUNOCHEMICAL STUDIES ON RHODOTORULA GRACILIS D-AMINO ACID OXIDASE

Polyclonal antibodies were prepared from rabbit sera after immunization with holo- and apo-D-amino acid oxidase purified fromR. gracilis. Both anti-holo- and anti-apoenzyme IgG fractions (as well as affinity-purified IgG) were highly specific: in blot-transfer analyses after SDS-PAGE only a 39 kDa band, corresponding to enzyme monomer, was recognized even in the partially purified yeast extract. No cross-reaction was detected with pig kidney D-amino acid oxidase. As a difference from the mammalian enzyme, yeast D-amino acid oxidase anti-holo- and anti-apoenzyme IgGs had different properties in inactivation and precipitation experiments, indicating the existence of different antigenicity sites related to the FAD-binding domain in the enzyme.

Stabilization of Yeast D-Amino Acid Oxidase by Matrix Covalent Attachment

Abstract D-amino acid oxidase from the yeast Rhodotorula gracilis has been immobilized onto Affi-Gel 10 matrix with a 60% yield under optimal conditions and a specific activity comparable to that of the free suspended preparation. No loss of FAD was observed in all experimental conditions; the enzyme-matrix complex exhibited a marked enhancement of stability against inactivation on heating, on organic solvents and on denaturant agents. Activation energies of the thermoinactivation process were 145.2 kJ/mol and 239.0 kJ/mol for immobilized and free form respectively. The thermal stabilization of the immobilized DAAO indicates that a more rigid molecular structure, stabilized against unfolding, resulted from the multipoint attachment of the protein to the support. The coupled system we have developed represents the first case where a marked increase of D-amino acid oxidase stability was obtained by matrix covalent attachment.