Stepwise in vitro synthesis of the dioxane unit in alnumycin A. The enzymatic reactions are drawn in black, and control reactions with boiled enzymes are in gray in the individual chromatograms shown at 470 nm. The reactions are from top to bottom. (A) Control with boiled AlnA, control with boiled AlnB, enzymatic synthesis of alnumycin C (5) from 1 mM d-ribulose-5-phosphate and 0.3 mM prealnumycin (2) by 30 μM AlnA and 10 μM AlnB (3.5 h incubation), and enzymatic synthesis of 5 with d-ribose-5-phosphate in place of d-ribulose-5-phosphate. (B) Control reaction with boiled Aln6, enzymatic synthesis of alnumycin B (4) from 0.3 mM 5 by 3 μM Aln6 (aerobic 1 h incubation). (C) Control reaction with boiled Aln4, enzymatic synthesis of alnumycin A (1), and alnumycin S (7) from 0.3 mM 4 and 4 mM NADPH by 15 μM Aln4 (90 min incubation).