Discovery of an L-fucono-1,5-lactonase from cog3618 of the amidohydrolase superfamily

Abstract

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 1

Metabolism of L-fucose. Pathway I produces dihydroxyacetone phosphate and L-lactaldehyde. Pathway II produces pyruvate and L-lactate through the oxidation of L-fucose to L-fucono-1,5-lactone.

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 or smaller. (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 or smaller. The nodes are color coded as follows: predicted L-fucono-1,5-lactonase (yellow nodes), BmulJ_04915 (blue square), Bamb_1224 (blue circle), Patl_0798 (blue triangle), L-rhamnono-1,4-lactonase (orange node), LigI (red node) and 4SML (green node).

Figure 3

Genomic neighborhood of BmulJ_04915. Genes are color coded as follows: cloned and purified for this study (red); predicted from strong sequence similarity to genes encoding proteins of known function (blue); predicted genes based on genomic context (yellow); predicted to function in carbohydrate transport (grey).

Figure 4

Structure of BmulJ_04915. (A) Ribbon diagram of BmulJ_04915. HEPES molecule shown as spheres. (B) Residues directly adjacent to bound HEPES in the active site of BmulJ_04915. Numbers in parenthesis designate the approximate location in the secondary structure from which the residue originates.

Figure 5

¹H-NMR time course for the non-enzymatic 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-(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 was adjusted to pH 6.5 the L-fucono-1,4-lactone is of equal concentration to that of the original enzymatic product. (C) 60 minutes after the pH of the reaction was 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-(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 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 non-enzymatic 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 β-anomer has been reduced to at least half of the original concentration.

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-(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 8

Ribbon diagram of BmulJ_04919 with bound NADP (magenta sticks) and L-fucose (green sticks).

Figure 9

Interactions of BmulJ-04919 with ligands. (A) 2.5σ F_o-F_c 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 stick with orange carbons, and residues of BmulJ_04919 adjacent to the 2′-adenosine phosphate are shown as sticks with green carbons. (D) 2.5σ F_o-F_c 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) Stereo view of the interactions of L-fucose with BmulJ_04919. Proteins 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 parenthesis designate the approximate location in the secondary structure from which the residue originates. (B) L-Fucono-1,5-lactone modeled into the active site of BmulJ_04915.

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