Structure-based mutational studies of substrate inhibition of betaine aldehyde dehydrogenase BetB from Staphylococcus aureus

Abstract

Inhibition of enzyme activity by high concentrations of substrate and/or cofactor is a general phenomenon demonstrated in many enzymes, including aldehyde dehydrogenases. Here we show that the uncharacterized protein BetB (SA2613) from Staphylococcus aureus is a highly specific betaine aldehyde dehydrogenase, which exhibits substrate inhibition at concentrations of betaine aldehyde as low as 0.15 mM. In contrast, the aldehyde dehydrogenase YdcW from Escherichia coli, which is also active against betaine aldehyde, shows no inhibition by this substrate. Using the crystal structures of BetB and YdcW, we performed a structure-based mutational analysis of BetB and introduced the YdcW residues into the BetB active site. From a total of 32 mutations, those in five residues located in the substrate binding pocket (Val288, Ser290, His448, Tyr450, and Trp456) greatly reduced the substrate inhibition of BetB, whereas the double mutant protein H448F/Y450L demonstrated a complete loss of substrate inhibition. Substrate inhibition was also reduced by mutations of the semiconserved Gly234 (to Ser, Thr, or Ala) located in the BetB NAD(+) binding site, suggesting some cooperativity between the cofactor and substrate binding sites. Substrate docking analysis of the BetB and YdcW active sites revealed that the wild-type BetB can bind betaine aldehyde in both productive and nonproductive conformations, whereas only the productive binding mode can be modeled in the active sites of YdcW and the BetB mutant proteins with reduced substrate inhibition. Thus, our results suggest that the molecular mechanism of substrate inhibition of BetB is associated with the nonproductive binding of betaine aldehyde.

FIG 1

Reaction catalyzed by S. aureus BetB and proposed mechanism. (A) NAD⁺-dependent oxidation of betaine aldehyde to glycine betaine. (B) Ordered Bi Bi mechanism proposed for BetB.

FIG 2

Dehydrogenase activity of BetB (A) and YdcW (B) as a function of BA concentration. Velocity (v) versus substrate concentration curves for S. aureus BetB (A) and E. coli YdcW (B). The dehydrogenase activity was determined using varied concentrations of BA, 5 mM NAD⁺, and 1 μg of BetB (10 μg for YdcW). Results are means ± standard deviations (SD) from at least two independent experiments.

FIG 3

Kinetic studies of BetB. (A) Initial velocity patterns of the BetB reaction. Dehydrogenase activity of BetB was determined with varied NAD⁺ (0.04 to 1.25 mM) in the presence of fixed concentrations of BA: 0.16 mM (diamonds), 0.31 mM (open squares), 0.62 mM (closed squares), 1.25 mM (open triangles), 2.5 mM (closed triangles), 5 mM (open circles), and 10 mM (closed circles). (B, C) Inhibition patterns of BetB activity by NADH. (B) Dehydrogenase activity of BetB as a function of NAD⁺ concentration in the presence of various concentrations of NADH: 2.5 μM (closed circles), 25 μM (open circles), 50 μM (closed triangles), and 100 μM (open triangles). BA concentration was 0.15 mM. The control (0 μM NADH) has the same line as 2.5 μM NADH. (C) Dehydrogenase activity of BetB as a function of BA concentration in the presence of various concentrations of NADH: 100 μM (closed circles), 250 μM (open circles), 375 μM (closed triangles), and 500 μM (open triangles). The control (0 μM NADH) has the same line as 100 μM NADH.

FIG 4

Active sites of BetB and YdcW. (A, B) Substrate binding pockets of BetB (A) and YdcW (B): a close-up view. The amino acid side chains and ligands are shown as sticks and labeled along with the protein ribbon. The residues of the double mutant protein H448F/Y450L are labeled in red. (C, D) Cofactor binding sites of BetB (C) and YdcW (D): a close-up view. The amino acid side chains and cofactors (NAD⁺) are shown as sticks and labeled along with the protein ribbon.

FIG 5

Activity profiles of the wild-type and mutant BetB proteins. Dehydrogenase activity was determined in the presence of 2 mM BA, 5 mM NAD⁺, and 1 μg protein. Results are means ± SD from at least two independent experiments. v, velocity.

FIG 6

Dehydrogenase activity of the wild-type and mutant BetB proteins as a function of BA concentration. (A) Wild-type BetB; (B) S290T; (C) Q162M; (D) G234T; (E) H448F/Y450L; (F) W456H. Reaction mixtures contained 5 mM NAD⁺ and 1 to 5 μg of enzyme. Results are means ± SD from at least two independent experiments. v, velocity.

FIG 7

Structural basis of BetB inhibition by BA: productive and nonproductive binding of BA. Docking simulation of BA binding in the active sites of wild-type BetB (A, B), YdcW (C), and the homology model of the double mutant protein H448F/Y450L (D). Panels A, C, and D represent the productive binding mode of BA, with a hydrogen bond formed between the carbonyl group of BA and the backbone nitrogen atom of Cys289 (in BetB). In contrast, panel B presents BA in a nonproductive binding mode with similar binding affinity and a hydrogen bond formed between the carbonyl group of BA and the amide group of Asn157. The amino acid side chains and ligands are shown as sticks and labeled. The residues of the double mutant protein H448F/Y450L are labeled in a larger font.