FIGURE 6 - Time-dependent synthesis of tyrosine and 5-hydroxytryptophan by K08F8.4 fusion protein (MBP-AH; abbreviated AH in figure) in vitro.
Reactions containing crude lysates from induced bacteria incubated with substrate and cofactor displayed hydroxylase activity. X-axis shows arbitrary units of fluorescence.
(A) Synthesis of tyrosine from phenylalanine increased over time and did not occur in reactions lacking cofactor, or in which lysate was boiled prior to incubation. Crude lysates from induced parental bacteria in complete reaction mix did not synthesize tyrosine. Symbols: open circle (AH + D) - Complete reaction with MBP-AH fusion protein, substrate and DMPH4 cofactor; open square (AH* + D) - complete reaction with heat-inactivated protein; open triangle (AH - D) - reaction without cofactor; closed circle (MBP + D) - complete reaction using lysate from induced parental bacteria expressing MBP protein alone.
(B) Synthesis of 5-HTP from tryptophan by MBP-AH protein. Symbols are as in part (A) Complete reaction done in triplicate; shown are means ± SEM of time points.