Abstract
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.
Highlights
Glycine oxidase (GO,1 EC 1.4.3.19) is a flavoprotein consisting of four identical subunits (369 residues each) and containing one molecule of noncovalently bound FAD per 42-kDa protein molecule (1, 2)
The most significant differences between the glycine oxidase (GO) structure and both MSOX and RgDAAO are represented by ␣-helix 8, which is missing in the other two enzymes, a different ␣-helix 3 and 4 topology, which is fused to a single continuous helix in RgDAAO and MSOX, and by the three stranded -sheet of the flavin-binding domain, which is conserved in all glutathione reductase (GR) family members and is not conserved in RgDAAO (10, 11)
On the basis of the data presented here, we propose that the structure of B. subtilis GO resembles that observed for MSOX and DAAO and that both enzymes have a catabolic role, in the specific mode of FAD and substrate binding different amino acids are involved
Summary
Growth Conditions and Preparation of Cell Extracts—B. subtilis pre-culture was grown aerobically at 37 °C in the dark and under shaking (180 rpm) on a chemically defined, pH-controlled liquid medium (minimal medium) containing 1ϫ minimal salt solution, 0.4% glucose, 0.005% L-tryptophan, 0.2% L-glutamine, 4 mg/ml FeCl3, 0.2 mg/ml MnSO4, and 1% (v/v) trace element salt solution. The 1ϫ trace element salt solution contained 43 M CaCl2, 12.5 M ZnCl2, 2.5 M CuCl2, 2.5 M CoCl2, and 2.5 M NaMoO4 This pre-culture was diluted to a final A600 ϳ 0.08 in 500 ml of minimal medium (2-liter flasks) and grown for 16 h as reported above. The cells were grown in flasks as reported above and collected at different growth phases by centrifugation (4000 rpm for 10 min at room temperature) from 100 ml of fermentation broth. GO crystals of the space group P6122 were obtained by using a reservoir solution containing 1 M sodium citrate, pH 6.2; the hexagonal crystals grew in 2–3 weeks. Heavy atom derivatives were prepared by addition in the reservoir buffer of KAu(CN) or K2Pt(CN) to the hexagonal crystal form of GO (1 mM final concentration) and incubating them for 12–16 h. Accession Numbers—The coordinates and structure factors of glycine oxidase in complex with the inhibitor glycolate have been deposited in the Protein Data Bank under accession code 1RYI
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