Structure-Based Mutational Studies of Substrate Inhibition of Betaine Aldehyde Dehydrogenase BetB from Staphylococcus aureus

Abstract

c 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. Inhibition of enzyme activity at high concentrations of substrate and/or cofactor is a general phenomenon observed in over 20% of known enzymes, including dehydrogenases, kinases, methyltransferases, and hydroxylases (1-3). Presently, it is considered to be a biologically relevant regulatory mechanism with important biological functions in several metabolic pathways (2). For example, substrate inhibition of tyrosine hydroxylase stabilizes the level of dopamine despite large changes in the tyrosine concentration, whereas the inhibition of acetylcholinesterase enhances the neural signal (2). However, substrate inhibition has a negative effect on biotechnological application of enzymes, because it reduces the reaction rate and product yield in industrial processes, which are usually performed at high substrate concentrations (4). For example, the inhibition of ␤-galactosidases by glucose and galactose limits the production of galacto-oligosaccharides (5).For dehydrogenases, several molecular mechanisms of substrate inhibition have been proposed, including the formation of a covalent adduct between the oxidized forms of substrate and cofactor, allosteric inhibition (which occurs away from the active site, e.g., in D-3-phosphoglycerol dehydrogenase from Mycobacterium tuberculosis), and the formation of a nonproductive enzyme complex with cofa...

Crystal structures of human 17β‐hydroxysteroid dehydrogenase type 1 complexed with estrone and NADP+ reveal the mechanism of substrate inhibition

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Protein‐inorganic calcium‐phosphate supraparticles as a robust platform for enzyme co‐immobilization

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Two-Step Production of Phenylpyruvic Acid from L-Phenylalanine by Growing and Resting Cells of Engineered Escherichia coli: Process Optimization and Kinetics Modeling

“…mirabilis [30] and amino acid oxidases from Rhodococcus opacus [31] and Pseudomonas putida [32]. The removal of substrate inhibition using the protein engineering techniques has been successfully used in many enzymes such as l -lactate dehydrogenase from Bacillus stearothermophilus [33], β-N-acetyl-D-hexosaminidases from Ostrinia furnacalis [34], and betaine aldehyde dehydrogenase from Staphylococcus aureus [35]. Due to the low similarity (19%) of l -AAD to other l -amino amino oxidases, it is difficult to develop a homology model to eliminate the substrate and product inhibition by directional protein engineering.…”