FIG 4.
Activities of LcpK30 muteins. (A) Activity of purified LcpK30 variants was determined by HPLC-based analysis of the cleavage products. For Lcp R328A, the typical wild-type pattern of oligoisoprenoids varying in the number of isoprene units (n) was observed (black line). For comparison, purified RoxA from Xanthomonas sp. 35Y producing 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al (ODTD) (n = 2) as sole major degradation product was also used (red line). For the wild type and most Lcp muteins, 4 μg of purified Lcp and a 2-h assay time were applied. For LcpK30 T168A (green line), an increased amount of enzyme (50 μg) and a longer incubation time (16 h) were used before the cleavage products were extracted with ethyl acetate and analyzed by HPLC. Note the very small amounts of degradation products with the same retention times and pattern produced by the T168A mutein. For data for the LcpK30 wild type and other Lcp muteins, see Fig. S4 in the supplemental material. (B) Assay of Lcp activity via the monitoring of oxygen consumption. After linearity of the oxygen consumption (3 min), the respective enzymes were added (arrow), initiating a decrease in oxygen saturation due to polyisoprene cleavage. Two parallel runs were recorded for the R328A mutein (red and green lines). Blank refers to a control without enzyme. For data for the other Lcp muteins see Fig. S3 in the supplemental material.