Members in the AH Bridging Project, Protein Core, and Structure Core determined that an EFI AH superfamily target catalyzes the hydrolysis of a series of sugar lactones. This is a novel function that further expands the AH superfamily catalog of reactions. This success provides additional support for the EFI’s large scale computation-based strategy for functional prediction.
A member of the amidohydrolase superfamily, BmulJ_04915 from Burkholderia multivorans, of unknown function was determined to hydrolyze a series of sugar lactones: L-fucono-1,4-lactone, D-arabino-1,4-lactone, L-xylono-1,4-lactone, D-lyxono-1,4-lactone, and L-galactono-1,4-lactone. The highest activity was shown for L-fucono-1,4-lactone with a k(cat) value of 140 s(-1) and a k(cat)/K(m) value of 1.0 × 10(5) M(-1) s(-1) at pH 8.3. The enzymatic product of an adjacent L-fucose dehydrogenase, BmulJ_04919, was shown to be L-fucono-1,5-lactone via nuclear magnetic resonance spectroscopy. L-Fucono-1,5-lactone is unstable and rapidly converts nonenzymatically to L-fucono-1,4-lactone. Because of the chemical instability of L-fucono-1,5-lactone, 4-deoxy-L-fucono-1,5-lactone was enzymatically synthesized from 4-deoxy-L-fucose using L-fucose dehydrogenase. BmulJ_04915 hydrolyzed 4-deoxy-L-fucono-1,5-lactone with a k(cat) value of 990 s(-1) and a k(cat)/K(m) value of 8.0 × 10(6) M(-1) s(-1) at pH 7.1. The protein does not require divalent cations in the active site for catalytic activity. BmulJ_04915 is the second enzyme from cog3618 of the amidohydrolase superfamily that does not require a divalent metal for catalytic activity. BmulJ_04915 is the first enzyme that has been shown to catalyze the hydrolysis of either L-fucono-1,4-lactone or L-fucono-1,5-lactone. The structures of the fuconolactonase and the fucose dehydrogenase were determined by X-ray diffraction methods.
Figure 2.(A) cog3618 sequence similarity networks at an E value of 10–30 where each node represents a protein and an edge represents an E value between two proteins of ≤10–30. (B) cog3618 sequence similarity networks at an E value of 10–70 where each node represents a protein and an edge represents an E value between two proteins of ≤10–70. The nodes are color-coded as follows: yellow nodes for predicted l-fucono-1,5-lactonase, blue square for BmulJ_04915, blue circle for Bamb_1224, blue triangle for Patl_0798, orange node for l-rhamnono-1,4-lactonase, red node for LigI, and green node for Protein Data Bank entry 4SML.
Figure 3. Genomic neighborhood of BmulJ_04915. Genes are color-coded as follows: red for those cloned and purified for this study, blue for those predicted from strong sequence similarity to genes encoding proteins of known function, yellow for those predicted genes based on genomic context, and gray for those predicted to function in carbohydrate transport.
Figure 4. Structure of BmulJ_04915. (A) Ribbon diagram of BmulJ_04915. The HEPES molecule is shown as spheres. (B) Residues directly adjacent to bound HEPES in the active site of BmulJ_04915. Numbers in parentheses designate the approximate location in the secondary structure from which the residue originates.
Figure 5. 1H NMR time course for the nonenzymatic conversion of l-fucono-1,5-lactone to l-fucono-1,4-lactone. The resonances corresponding to the C-6 methyl groups of both α-l-fucose (1.12 ppm) and β-l-fucose (1.15 ppm), l-fucono-1,5-lactone (1.32 ppm), and l-fucono-1,4-lactone (1.24 ppm) are presented. (A) The major reaction product of BmulJ_04915 at pH 4.2 is shown to be l-fucono-1,5-lactone. (B) Five minutes after the reaction mixture had been adjusted to pH 6.5, the l-fucono-1,4-lactone is at a concentration equal to that of the original enzymatic product. (C) Sixty minutes after the pH of the reaction mixture had been adjusted to pH 6.5, the major peak is l-fucono-1,4-lactone.
Figure 6. 1H NMR time course of the enzymatic conversion of l-fucose to l-fucono-1,5-lactone and l-fucono-1,5-lactone to l-fuconate. The resonances corresponding to the C-6 methyl groups of both α-l-fucose (1.125 ppm) and β-l-fucose (1.15 ppm), l-fucono-1,5-lactone (1.295 ppm), l-fucono-1,4-lactone (1.225 ppm), and l-fuconate (1.182 ppm) are provided. (A) l-Fucose, the substrate for BmulJ_04919, prior to addition of the enzyme at pH 6.5. (B) Five minutes after the addition of BmulJ_04919 to l-fucose, the enzymatic product, l-fucono-1,5-lactone, and the nonenzymatic product, l-fucono-1,4-lactone, are at equal concentrations. (C) Five minutes after the addition of BmulJ_04915 to the reaction mixture, the l-fucono-1,5-lactone is no longer present in the reaction mixture and the l-fucono-1,4-lactone appears to be unchanged. l-Fuconate (1.182 ppm) appears to be the major product. The α-anomer of l-fucose appears to be unchanged; however, the concentration of the β-anomer has been reduced to at least half of the original value.
Figure 7. 1H NMR time course of the enzymatic conversion of 4-deoxy-l-fucose to 4-deoxy-l-fucono-1,5-lactone and 4-deoxy-l-fuconate. The resonances corresponding to the C-6 methyl groups of both α-4-deoxy-l-fucose (1.02 ppm) and β-4-deoxy-l-fucose (1.06 ppm), 4-deoxy-l-fucono-1,5-lactone (1.25 ppm), and 4-deoxy-l-fuconate (1.07 ppm) are provided. (A) 4-Deoxy-l-fucose, prior to addition of enzymes at pH 6.5. (B) One minute after the addition of BmulJ_04919 to 4-deoxy-l-fucose, the enzymatic product is 4-deoxy-l-fucono-1,5-lactone. (C) One minute after the addition of BmulJ_04915 to the reaction mixture, 4-deoxy-l-fucono-1,5-lactone is no longer present in the reaction mixture and 4-deoxy-l-fuconate (1.07 ppm) appears to be the major product.
Figure 9. Interactions of BmulJ_04919 with ligands. (A) The 2.5σ Fo – Fc kick map for NADP+ bound to the NADP+–l-fucose–BmulJ_04919 ternary complex. (B) Interactions of NADP+ with the secondary structure elements of BmulJ_04919. (C) 2′-Adenosine phosphate binding site of BmulJ_04919. NADP+ is shown as sticks with orange carbons, and residues of BmulJ_04919 adjacent to the 2′-adenosine phosphate are shown as sticks with green carbons. (D) The 2.5σ Fo – Fc kick map for l-fucose bound to the NADP+–l-fucose–BmulJ_04919 ternary complex. l-Fucose is shown as sticks with yellow, numbered carbons. (E) Stereoview of the interactions of l-fucose with BmulJ_04919. Protein atoms, l-fucose, and NADP+ are shown as sticks with green, yellow, and white carbons, respectively.
Figure 10. (A) Active site of BmulJ_04915 structurally aligned with that of 2-pyrone-4,6-dicarboxylic acid lactonase (LigI). The active site is color-coded as follows: green for LigI (PDB entry 4d8l) and white for BmulJ_04915. Numbers in parentheses designate the approximate locations in the secondary structure from which the residue originates. (B) l-Fucono-1,5-lactone modeled into the active site of BmulJ_04915.
Reprinted with permission from the Journal of Biochemistry.
© 2013 American Chemical Society.