Design and properties of human D-amino acid oxidase with covalently attached flavin.

An "artificial flavinylation" approach was developed to replace a native noncovalent flavin prosthetic group with a covalently attached flavin analogue in recombinant human d-amino acid oxidase. The protein residue Gly-281 was replaced with Cys by site-directed mutagenesis, followed by reaction between mutated apoenzyme and the thiol-reactive flavin analogue, 8-methylsulfonyl FAD. The stoichiometric process of flavin attachment was accompanied by gain in enzymatic activity, reaching up to 26% activity of the recombinant native enzyme. The steady-state kinetic data together with the results of limited proteolysis and benzoate-binding studies suggest that, although mutation perturbs protein structural and catalytic properties, the flavinylation alone does not have any negative impact. We conclude that, despite the implemented restraints on its mobility, the covalently attached flavin is properly positioned within the protein active site and acts efficiently during d-amino acid oxidase catalytic turnover.

Arabidopsis NPH1: a flavoprotein with the properties of a photoreceptor for phototropism.

The NPH1 gene of Arabidopsis thaliana encodes a 120-kilodalton serine-threonine protein kinase hypothesized to function as a photoreceptor for phototropism. When expressed in insect cells, the NPH1 protein is phosphorylated in response to blue light irradiation. The biochemical and photochemical properties of the photosensitive protein reflect those of the native protein in microsomal membranes. Recombinant NPH1 noncovalently binds flavin mononucleotide, a likely chromophore for light-dependent autophosphorylation. The fluorescence excitation spectrum of the recombinant protein is similar to the action spectrum for phototropism, consistent with the conclusion that NPH1 is an autophosphorylating flavoprotein photoreceptor mediating phototropic responses in higher plants.

Primary structure of the snake venom L-amino acid oxidase shows high homology with the mouse B cell interleukin 4-induced Fig1 protein.

The 2.8-kb sequence of cDNA encoding southeastern diamondback rattlesnake venom L-amino acid oxidase was determined. A translated single open reading frame revealed a 58.7-kDa polypeptide containing a signal sequence and FAD binding motif. The protein similarity search and analysis showed that the snake L-amino acid oxidase possesses the highest degree of homology (37% of identity) with a protein encoded by the mouse B cell interleukin 4-induced Fig1 gene.

Glycerol-assisted restorative adjustment of flavoenzyme conformation perturbed by site-directed mutagenesis.

The replacement of histidine 307 with leucine in pig kidney D-amino acid oxidase perturbs its active site conformation accompanied by dramatic losses in protein-flavin interactions and enzymatic activity. However, the negative effect of this mutation on the holoenzyme structure is essentially eliminated in the presence of glycerol, resulting in up to 50% activity recovery and greater than 16-fold increase in the flavin affinity. Further analysis revealed that glycerol assists in the rearrangement of the protein toward its holoenzyme-like conformation together with reduction in the solvent-accessible protein hydrophobic area as demonstrated by limited proteolysis and use of affinity and hydrophobic probes. A substantial decrease in the protein-flavin interactions was demonstrated at a low temperature, but this reversible process was completely blocked in the presence of 40% glycerol. We suggest that the perturbation of the D-amino acid oxidase active site is due to the nonpolar nature of the mutation whose negative impact on the holoenzyme structure can be overcome by glycerol-induced strengthening of protein internal hydrophobic interactions.

Identification of a reactive cysteine in the flavin-binding domain of Rhodotorula gracilis D-amino acid oxidase.

The holoenzyme form of Rhodotorula gracilis D-amino acid oxidase, an 80-kDa homodimer, reacted only to a limited extent with general thiol reagents (2,2'-dithiodipyridine, 5,5'-dithiobis(2-nitrobenzoic acid), and N-[7-(dimethylamino)-4-methylcoumarinyl]maleimide) (60% residual activity), whereas the monomeric apoprotein was completely inactivated and denatured by these reagents. To investigate the presence of thiol residue(s) in the active site of the enzyme, the apoprotein was reconstituted with the 8-(methylsulfonyl)-FAD chemical-affinity probe. Competitive inhibition between this analogue and FAD for apoprotein binding was observed. The covalent attachment of the flavin analogue to the apoprotein was complete after approximately 20 h of incubation and the flavinylated enzyme, containing 8-(cysteinyl)-FAD, was monomeric and inactive. After HPLC isolation of the flavin-labeled tryptic peptides, Cys208 was identified as the only cysteine to react with the FAD analogue. These results show that a single cysteine of R. gracilis D-amino acid oxidase reacts with the flavin analogue and that this is located near or at the FAD-binding domain.

Glycerol-induced development of catalytically active conformation of Crotalus adamanteus L-amino acid oxidase in vitro.

The reconstitutable apoprotein of Crotalus adamanteus L-amino acid oxidase was prepared using hydrophobic interaction chromatography. After reconstitution with flavin adenine dinucleotide, the resulting protein was inactive, with a perturbed conformation of the flavin binding site. Subsequently, a series of cosolvent-dependent compact intermediates was identified. The nearly complete activation of the reconstituted apoprotein and the restoration of its native flavin binding site was achieved in the presence of 50% glycerol. We provide evidence that in addition to a merely stabilizing effect of glycerol on native proteins, glycerol can also have a restorative effect on their compact equilibrium intermediates, and we suggest the hydrophobic effect as a dominating force in this in vitro-assisted restorative process.

Association of flavin adenine dinucleotide with the Arabidopsis blue light receptor CRY1.

The arabidopsis thaliana HY4 gene encodes CRY1, a 75-kilodalton flavoprotein mediating blue light-dependent regulation of seedling development. CRY1 is demonstrated here to noncovalently bind stoichiometric amounts of flavin adenine dinucleotide (FAD). The redox properties of FAD bound by CRY1 include an unexpected stability of the neutral radical flavosemiquinone (FADH.). The absorption properties of this flavosemiquinone provide a likely explanation for the additional sensitivity exhibited by CRY1-mediated responses in the green region of the visible spectrum. Despite the sequence homology to microbial DNA photolyases, CRY1 was found to have no detectable photolyase activity.

Affinity probing of flavin binding sites. 1. Covalent attachment of 8-(methylsulfonyl)FAD to pig heart lipoamide dehydrogenase.

8-(Methylsulfonyl)FAD reacts with a single cysteine residue (Cys449) in pig apolipoamide dehydrogenase to generate a flavinylated enzyme containing covalently bound 8-(cysteinyl)FAD. Competitive behavior is observed in reconstitution reactions containing both FAD and 8-(methylsulfonyl)FAD. Covalently bound 8-(cysteinyl)FAD is shielded from solvent, as judged by spectral comparison with model 8-(alkylthio)-flavins in various solvents. Flavinylated lipoamide dehydrogenase is monomeric and catalytically inactive. Cys449 is located in the interface domain, near the active site histidine (His452). As shown previously, Cys449 is oxidized when native enzyme is treated with cupric ions. Cys449 is close to the isoalloxazine ring of FAD in native enzyme, as judged by alignment of the pig sequence with the structure of the homologous enzyme from Azotobacter vinelandii. The residue corresponding to Cys449 in A. vinlandii lipoamide dehydrogenase (Val447) is about 9 A from the carbonyl oxygen at C(2) in the pyrimidine ring of FAD. Approximation of a substituent at position 8 in FAD with Cys449 requires a 180 degrees flip of the isoalloxazine ring as compared with its orientation in the native structure. The different flavin orientation can explain the absence of dimerization and catalytic activity. Using the same method of apoenzyme preparation, noncovalent binding was observed with 8-chloroFAD, a less reactive flavin analogue. Relatively nonspecific covalent incorporation was observed with 8-chloroFAD when apoenzyme was prepared by an older method used in previous studies with this derivative [Moore, E.G., Cardemil, E., & Massey, V. (1978) J. Biol. Chem. 253, 6413-6422].

Affinity probing of flavin binding sites. 2. Identification of a reactive cysteine in the flavin domain of Escherichia coli DNA photolyase.

8-(Methylsulfonyl)FAD reacts with a single cysteine residue (Cys293) in the flavin domain of Escherichia coli DNA photolyase to form an 8-(cysteinyl)FAD derivative covalently bound to the protein. About 80% protection against covalent attachment with 8-(methylsulfonyl)FAD was observed in the presence of an equimolar amount of FAD. Flavinylated photolyase retains the ability to repair pyrimidine dimers (15% of native activity) and to bind its antenna chromophore, 5,10-methenyltetrahydrofolate. Comparison of the properties of flavinylated enzyme with photolyase containing noncovalently bound 8-(methylthio)-FAD indicate that a perturbation is necessary to accommodate covalent bond formation. 8-(Methylthio)-FAD-reconstituted enzyme exhibits 95% of native activity. The aerobic stability of fully reduced and radical forms of 8-(methylthio)FAD enzyme is similar to that of native enzyme, whereas a radical form is not detected with flavinylated enzyme and the fully reduced enzyme is more easily oxidized by oxygen. The flavin in 8-(methylthio)FAD enzyme or flavinylated photolyase is shielded from solvent. However, the flavin environment in flavinylated enzyme is less hydrophobic as judged by spectral comparison with model 8-(alkylthio)flavins in various solvents. Enzyme containing noncovalently bound 8-(methylsulfonyl)-FAD was prepared by reconstitution with the fully reduced flavin which does not undergo covalent attachment. Covalent attachment was observed after reoxidation but probably involved dissociation and rebinding of oxidized 8-(methylsulfonyl)FAD. The results show that 8-(cysteinyl)FAD in flavinylated photolyase is at or near the normal flavin binding site.(ABSTRACT TRUNCATED AT 250 WORDS)

Properties of a high-potential flavin analogue and its use as an active site probe with clostridial flavodoxin.

The reduction potential of flavin bearing a methylsulfonyl moiety (MeSO2) in place of a methyl group at position 8 is increased by more than 150 mV as compared with normal flavin. This substitution is accompanied by a substantial increase in reactivity with various reductants, including NADH, and greatly (10(3)-fold) enhanced susceptibility toward nucleophilic attack by sulfite at N(5). 1,5-Dihydro-8-(methylsulfonyl)riboflavin exhibits two intense, well-resolved absorption bands (lambda max = 310, 362 nm) in a region where most other reduced flavins exhibit weak, characterless absorption. This unusual spectrum is attributable to a shift of pi-electron density from the N(5) atom into the benzene ring. It is observed only with reduced flavins bearing a strongly electronegative substituent (MeSO2, CN) at the 8-position. The effect is abolished by replacing the hydrogen at N(5) with a bulky group, like sulfite, which interferes with sp2 hybridization at N(5). Reaction of 8-MeSO2-substituted flavins with thiols results in nucleophilic displacement of MeSO2- in a reaction that is about 10(3)-fold faster than an analogous nucleophilic displacement reaction observed with 8-halo-substituted flavins. The flavin ring acts as a redox switch in controlling electrophilicity at the 8-position, as judged by the fact that the displacement reactions are observed only with the oxidized flavins. Initial studies to evaluate 8-MeSO2-substituted flavins as active site probes were conducted with flavodoxin from Clostridium beijerinckii MP. 8-MeSO2FMN is rapidly bound to apoflavodoxin, accompanied by absorbance and fluorescence changes similar to those observed for FMN binding. 1,5-Dihydro-8-MeSO2FMN flavodoxin exhibits spectral properties (lambda max = 323, 382 nm) similar to those of the corresponding free flavin, except for a bathochromic shift due to a change in the polarity of the flavin environment. As judged by peak resolution and intensity, the spectral properties of 1,5-dihydro-FMN flavodoxin (lambda max = 311, 362 nm) appear to lie about midway between those observed for the free 1,5-dihydro forms of FMN versus 8-MeSO2FMN. This suggests that the protein environment may favor enhanced resonance delocalization of pi-electron density into the benzene ring of bound 1,5-dihydro-FMN, as compared with the free flavin. This hypothesis is consistent with previous NMR studies and with a proposal that electron transfer from reduced flavodoxin to other redox proteins occurs through this region of the ring. 8-MeSO2FMN bound to flavodoxin reacts readily with exogenous thiols but does not react with sulfite.(ABSTRACT TRUNCATED AT 400 WORDS)

Green flavoprotein from P. leiognathi: purification, characterization and identification as the product of the lux G(N) gene.

A green flavoprotein (GFP) was isolated and purified to homogeneity from Photobacterium leiognathi, strain 208. GFP is a homodimer of molecular weight 54,000 and contains two molecules of an unusual flavin per molecule of protein. Various biochemical characteristics including isoelectric point, trypsin and chymotrypsin degradation, SDS and temperature influence on subunit dissociation and the dissociation of the flavin chromophore, were investigated. The sequence of 23 N-terminal amino acids was determined and found to be concurrent with the N-terminal amino acid sequence encoded by the lux G(N) gene of P. leiognathi. This fact suggests that GFP is a structural component of the Photobacterium luminescence system.