Fig. 2. Use of a TTA-ADH coupled assay for screening activity of ObiH on a diverse array of aromatic aldehyde substrates.

a Reaction schematic for coupled enzyme reaction that enables reaction monitoring at 340 nm if appropriate conditions and controls are used. Important negative controls are no addition of aldehyde (to account for the rate of l-Thr decomposition) and no addition of ObiH (to account for potential ADH-catalyzed reduction of the aldehyde substrate). b Initial rates of ObiH on aldehyde substrates relative to an l-Thr background measurement and ADH background activity on aldehydes. The horizontal line indicates the l-Thr background decomposition observed in the TTA-ADH coupled assay. Any activity greater than the dotted line and the corresponding ADH activity is considered a successful activity of the TTA on that aldehyde. The experiment was performed in triplicate with each replicate displayed as an individual data point and error bars representing standard deviations. c Chemical structures of the aldehydes investigated in this study. Asterisks indicate substrates never previously screened with TTAs to our knowledge.