FAD binding in glycine oxidase from Bacillus subtilis.

The apoprotein of the FAD-containing flavoenzyme glycine oxidase from Bacillus subtilis was obtained at pH 8.5 by dialyzing the holoenzyme against 2 M KBr in 0.25 M Tris-HCl and 20% glycerol. The apoprotein of glycine oxidase shows high protein fluorescence, high exposure of hydrophobic surfaces, and low temperature stability as compared to the holoenzyme. The isolated apoprotein species is present in solution as a monomer which rapidly recovers its tertiary structure and converts into the tetrameric holoenzyme following incubation with free FAD. The reconstitution process follows a particular two-stage process; the spectral properties of the reconstituted holoenzyme were virtually indistinguishable from those observed with native glycine oxidase, while the activity was only partially (50%) recovered. The urea-induced unfolding process of glycine oxidase can be considered as a two-step (three-state) process: the presence of intermediate(s) in the unfolding process of the holoenzyme at approximately 2 M urea is evident in the changes of the flavin fluorescence intensity and can be also inferred from the different urea sensitivities of the spectral probes used. On the other hand, only a single transition at approximately 4.5 M urea concentration is observed for the apoprotein form. The chemical denaturation of glycine oxidase holoenzyme is partially reversible (e.g., no activity is recovered when starting the refolding from 4 M urea-denatured holoprotein). Finally, the introduction by site-directed mutagenesis of residues corresponding to those involved in the covalent link with FAD in the related flavoenzyme monomeric sarcosine oxidase failed to convert glycine oxidase into a covalent flavoprotein. These investigations show that the consequences of FAD binding for the stability and folding process distinguish glycine oxidase from enzymes active on similar compounds.

Catalytic and redox properties of glycine oxidase from Bacillus subtilis.

Glycine oxidase (GO) from Bacillus subtilis is a homotetrameric flavoprotein oxidase that catalyzes the oxidation of the amine functional group of sarcosine or glycine (and some D-amino acids) to yield the corresponding keto acids, ammonia/amine and H(2)O(2). It shows optima at pH 7-8 for stability and pH 9-10 for activity, depending on the substrate. The tetrameric oligomeric state of the holoenzyme is not affected by pH in the 6.5-10 range. Free GO forms the anionic red semiquinone upon photoreduction. This species is thermodynamically stable, as indicated by the large separation of the two single-electron reduction potentials (DeltaE >or = 290 mV). The first potential is pH independent, while the second is dependent. The midpoint reduction potential exhibits a -23.4 mV/pH unit slope, which is consistent with an overall two-electrons/one-proton transfer in the reduction to yield anionic reduced flavin. In the presence of glycolate (a substrate analogue) and at pH 7.5 the potential for the semiquinone-reduced enzyme couple is shifted positively by approximately 160 mV: this favors a two-electron transfer compared to the free enzyme. Binding of glycolate and sulfite is also affected by pH, showing dependencies that reflect the ionization of an active site residue with a pK(a) approximately equal 8.0. These results highlight substantial differences between GO and related flavoenzymes. This knowledge will facilitate biotechnological use of GO, e.g. as an innovative tool for the in vivo detection of the neurotransmitter glycine.

Glycine oxidase from Bacillus subtilis: role of histidine 244 and methionine 261.

The reactions of several mutants at position 244 and 261 of bacterial glycine oxidase (GO) were studied by stopped-flow and steady-state kinetic methods. Substituting H244 with phenylalanine, glutamate, and glutamine and M261 with histidine and tyrosine did not affect the expression of GO and the physicochemical properties of bound FAD. All the H244 and M261 mutants of GO we prepared retained activity in both steady-state and stopped-flow kinetic studies, indicating they do not serve as key elements in glycine and sarcosine oxidation. We demonstrated that the substitution of H244 significantly affected the rate of flavin reduction with glycine even if this change did not modify the turnover number, which is frequently increased compared to wild-type GO. However, substitution of M261 affected the interaction with substrates/inhibitors and the rate of flavin reduction with sarcosine and resulted in a decrease in turnover number and efficiency with all the substrates tested. The considerable decrease in the rate of flavin reduction changed the conditions such that it was partially rate-limiting in the catalytic cycle compared to the wild-type GO. Our studies show some similarities, but also major differences, in the catalytic mechanism of GO and other flavooxidases also active on glycine and sarcosine and give insight into the mode of modulation of catalysis and substrate specificities.

Structure-function correlation in glycine oxidase from Bacillus subtilis.

Structure-function relationships of the flavoprotein glycine oxidase (GO), which was recently proposed as the first enzyme in the biosynthesis of thiamine in Bacillus subtilis, has been investigated by a combination of structural and functional studies. The structure of the GO-glycolate complex was determined at 1.8 A, a resolution at which a sketch of the residues involved in FAD binding and in substrate interaction can be depicted. GO can be considered a member of the "amine oxidase" class of flavoproteins, such as d-amino acid oxidase and monomeric sarcosine oxidase. With the obtained model of GO the monomer-monomer interactions can be analyzed in detail, thus explaining the structural basis of the stable tetrameric oligomerization state of GO, which is unique for the GR(2) subfamily of flavooxidases. On the other hand, the three-dimensional structure of GO and the functional experiments do not provide the functional significance of such an oligomerization state; GO does not show an allosteric behavior. The results do not clarify the metabolic role of this enzyme in B. subtilis; the broad substrate specificity of GO cannot be correlated with the inferred function in thiamine biosynthesis, and the structure does not show how GO could interact with ThiS, the following enzyme in thiamine biosynthesis. However, they do let a general catabolic role of this enzyme on primary or secondary amines to be excluded because the expression of GO is not inducible by glycine, sarcosine, or d-alanine as carbon or nitrogen sources.

Regulation of D-amino acid oxidase expression in the yeast Rhodotorula gracilis.

Rhodotorula gracilis is a oleaginous yeast which utilizes D-amino acids as a source of carbon and/or nitrogen. D-amino acid oxidase (DAAO), which converts D-amino acids in the corresponding alpha-keto acids and ammonia, is the first enzyme involved in the catabolism of D-amino acids. DAAO activity is induced by the presence of D-alanine, but the presence of the L-isomer prevents induction by inhibiting the transport of D-alanine into cells. To understand how DAAO expression is regulated, R. gracilis cells were grown on media containing different nitrogen and/or carbon sources. As a general rule, the level of DAAO mRNA reached a maximum after 15 h growth and preceded by approximately 6 h the maximum level of DAAO activity. The inducer D-alanine acts by increasing the rate of DAAO mRNA transcription: the increase in DAAO expression is due essentially to de novo synthesis. The presence of a supplemental carbon source (e.g. succinate or glucose) does not repress DAAO expression. Ammonium sulphate appears to have a negative effect on DAAO mRNA translation and on the expression of DAAO activity: DAAO is only partially active when the yeast is grown in the presence of D-alanine and ammonium sulphate. The best expression of DAAO activity was obtained by growing the cells for 12 h at 30 degrees C in the presence of glucose and D-alanine using cells pre-cultured for 10 h on glucose and L-alanine (0.99 U/mg protein, corresponding to approximately 1.0% total proteins in the crude extract). Under these growth conditions a six-fold increase in DAAO production was achieved.

Kinetic mechanisms of glycine oxidase from Bacillus subtilis.

The kinetic properties of glycine oxidase from Bacillus subtilis were investigated using glycine, sarcosine, and d-proline as substrate. The turnover numbers at saturating substrate and oxygen concentrations were 4.0 s(-1), 4.2 s(-1), and 3.5 s(-1), respectively, with glycine, sarcosine, and D-proline as substrate. Glycine oxidase was converted to a two-electron reduced form upon anaerobic reduction with the individual substrates and its reductive half-reaction was demonstrated to be reversible. The rates of flavin reduction extrapolated to saturating substrate concentration, and under anaerobic conditions, were 166 s(-1), 170 s(-1), and 26 s(-1), respectively, with glycine, sarcosine, and D-proline as substrate. The rate of reoxidation of reduced glycine oxidase with oxygen in the absence of product (extrapolated rate approximately 3 x 10(4) M(-1) x s(-1)) was too slow to account for catalysis and thus reoxidation started from the reduced enzyme:imino acid complex. The kinetic data are compatible with a ternary complex sequential mechanism in which the rate of product dissociation from the reoxidized enzyme form represents the rate-limiting step. Although glycine oxidase and D-amino acid oxidase differ in substrate specificity and amino acid sequence, the kinetic mechanism of glycine oxidase is similar to that determined for mammalian D-amino acid oxidase on neutral D-amino acids, further supporting a close similarity between these two amine oxidases.