Figure 6

Stereochemical preference of enzymes and product composition. (a) Relative AKR reaction rates for oxidation of various alcohols. AKR has a strong preference for (S) 1,3-BDO. Relative activity is the fractional rate compared to 2% (R) + 2% (S) 1,3-BDO. (b) DERA reaction rates using equal concentrations of the substrates (R) or (R,S) 3-HBal. We assessed DERA specificity in two phases. In the first phase, we generated (R) or (R,S) 3-HBal from 1,3-BDO using AKR and NADP⁺. In the second phase, we then measured the activity of DERA on the two different stereoisomeric populations of 3-HBal by the addition of DERA, ADH and NADH. In this second phase, DERA generates acetaldehyde, which is detected by ADH reduction to ethanol and concomitant oxidation of ethanol. The reduction of NADP+ in the first phase and the oxidation of NADH in the second phase can both be monitored by absorbance at 340 nm. The results indicate that (R) 3-HBal is the preferred substrate. (c) Scheme for assessing the stereochemical composition of the 1,3-BDO product of the DERA/AKR system. First, the 1,3-BDO product of unknown stereochemical composition was made from acetaldehyde using DERA and AKR enzymes as in the full enzyme system. Then the enzymes were removed and the concentration of the 1,3-BDO was determined (31 mM). To assess the stereochemical composition, we then measured the rate of the AKR catalyzed reaction back to 3-HBal. This rate was compared to the rates of the same AKR catalyzed reaction using substrates of known stereochemical composition (d). (d) The standard curve measuring the rate of the AKR catalyzed conversion of 31 mM 1,3-BDO to 3-HBal at different stereochemical compositions. Comparing to the known reaction rates to the rate obtained for the product of converting acetaldehyde to 1,3-BDO using DERA and AKR (red dot), allows us to estimate the stereochemical composition of the product at ~ 17% (S) 1,3 BDO. Error bars reflect standard deviations of biological triplicates.