Strategic manipulation of the substrate specificity of Saccharomyces cerevisiae flavocytochrome b2

Abstract

Flavocytochrome b2 from Saccharomyces cerevisiae acts physiologically as an L-lactate dehydrogenase. Although L-lactate is its primary substrate, the enzyme is also able to utilize a variety of other (S)-2-hydroxy acids. Structural studies and sequence comparisons with several related flavoenzymes have identified the key active-site residues required for catalysis. However, the residues Ala-198 and Leu-230, found in the X-ray-crystal structure to be in contact with the substrate methyl group, are not well conserved. We propose that the interaction between these residues and a prospective substrate molecule has a significant effect on the substrate specificity of the enzyme. In an attempt to modify the specificity in favour oflarger substrates, three mutant enzymes

The Ala95‐to‐Gly substitution in Aerococcus viridansl‐lactate oxidase revisited – structural consequences at the catalytic site and effect on reactivity with O2 and other electron acceptors

“…A naturally occurring Ala/Gly residue variation within strand b1 is a prominent factor of structural variability around the FMN N5 atom [3][4][5][6][7]. Evidence from sitedirected mutagenesis reveals that Ala/Gly residue exchange in different enzymes impacts significantly upon the O 2 reactivity, in addition to having a major effect on the a-hydroxy acid substrate specificity [19][20][21][22][23]. However, the results also show that the Ala/Gly pattern does not constitute a simply addressable oxidase-dehydrogenase switch in a-hydroxy acid-oxidizing flavoenzymes, unlike vanillyl-alcohol oxidases where a similar pattern of Ala and Gly (or Pro) was proposed to determine flavoenzyme activity as a dehydrogenase and oxidase, respectively [24].…”

Rationally re-designed mutation of NAD-independent l-lactate dehydrogenase: high optical resolution of racemic mandelic acid by the engineered Escherichia coli

“…The X-ray crystal structure of flavocytochrome b 2 has been resolved (PDB ID code 1FCB), and the active site for 2-α-hydroxyacid dehydrogenation has been identified [23]. Six amino acids which interact directly to the substrate have been pinpointed in the crystal structure [24], in which four amino acids are highly conserved in this protein family. However, the other two residues, Ala-198 and Leu-230 in flavocytochrome b 2 , which interact with the alkyl group of substrates, are not well conserved (Additional file 1 Figure S1).…”

Flavocytochrome b 2

“…The substitution of Ile326 by alanine, which removes substantial bulk from a position directly adjacent to Leu230, also shifted the selectivity of the enzyme towards longer chain substrates and particularly 2-hydroxyoctanoate (Daff et al, 1994b). A rather interesting observation was that, although the enzyme works well on straight-chain substrates and substrates which branch at the C-3 position, no activity was seen with substrates branched at C-4 (Daff et al, 1994a and1994b).…”

“…Sequence comparisons indicate that residues Ala198 and Leu230, highlighted in Figure 7, are not well conserved in these enzymes adding support to the idea that they are important in substrate selection. A shift in the selectivity of flavocytochrome b 2 , away from lactate and towards longer chain substrates, has been achieved by making substitutions of these residues by site-directed mutagenesis (Daff et al, 1994a and1994b). The substitution of Leu230 by alanine had a significant effect, causing the selectivity of the enzyme for 2-hydroxyoctanoate over lactate to increase by a factor of 80 (Daff et al, 1994a).…”

“…A shift in the selectivity of flavocytochrome b 2 , away from lactate and towards longer chain substrates, has been achieved by making substitutions of these residues by site-directed mutagenesis (Daff et al, 1994a and1994b). The substitution of Leu230 by alanine had a significant effect, causing the selectivity of the enzyme for 2-hydroxyoctanoate over lactate to increase by a factor of 80 (Daff et al, 1994a). The substitution of Ile326 by alanine, which removes substantial bulk from a position directly adjacent to Leu230, also shifted the selectivity of the enzyme towards longer chain substrates and particularly 2-hydroxyoctanoate (Daff et al, 1994b).…”