the substrate for the methylation reaction, and controls containing no substrate will therefore show little or no incorporation of label from [ 14CH~] S-adenosylmethionine.
 N A D P :Tetracycline 5a (I la)Dehydrogenase By PHILIP A. MILLER and JOHN H. HASH NADPH+H
7- Chloro-5a (1la)dehydrotetracycline
Enzymic reduction of the 5 a - l l a double bond has been shown to be the final reaction in the biosynthesis of the tetracycline group of antibiotics. 1,2 The reaction occurs in antibiotic-producing strains of Streptomyces aureo]aciens as well as in various blocked mutants derived therefrom. Of the various 5 a ( l l a ) - d e h y d r o t e t r a c y c l i n e s which have been prepared, only 7-chloro-5a(lla)-dehydrotetracycline is chemically stable; we have therefore chosen it as the substrate to illustrate the reaction described here. Assay
Principle. The presence of 5 a ( l l a ) dehydrogenase activity can be determined by measuring an increase in antibacterial activity due to the synthesis of 7-chlorotetracycline from its inactive precursor, 7-chloro5 a ( l l a ) - d e h y d r o t e t r a c y c l i n e . Any tetracycline-sensitive bacteria can be used, and either a turbidimetric or agar-diffusion type assay is suitable2 Reagents T r i s . H C l buffer, 0.2 M, pH 7.0 N A D P H , 2 m M in water 7-Chloro-5a(lla)-dehydrotetracycline, 1 m M in methanol 1p. A. Miller, J. H. Hash, M. Lincks, and N. Bohonos, Biochem. Biophys. Res. Commun. 18, 325 (1965). : P. A. Miller, Develop. Ind. Microbiol. 8, 96 (1967). "This volume .
NADP :TETRACYCLINE 5a(11 a)I:)EttYDROGENASE
Procedure. A reaction mixture is prepared by combining 0.1 ml of the substrate, 0.1 ml of NADPH, 0.3 ml of water, and 0.5 ml of a 20 mg/ml solution of lyophilized cell-free extract in Tris buffer. The reaction mixture is incubated at 28 ° for 2 hr and assayed for antibiotic activity using a Staphylococcus aureus turbidimetric method. ~ The product, 7-chlorotetracycline, can be identified using the following paper chromatographic system: Whatman No. 1 paper buffered with 0.3 M phosphate, pH 3.0, and developed with n-butanol saturated with water. Preparation o] 7-C hloro-5a (11a )-dehydrotetracycli~w. This substrate can be prepared by fermentation using a blocked mutant of S. aureofaciens, ATCC 12748. A vegetative inoculum of this strain is prepared by inoculating spores into a medium containing, in grams per liter: sucrose, 30; ammonium sulfate, 2; calcium carbonate, 7; and corn steep liquor, 16.5 ml. After incubation on a reciprocating shaker for 24 hr at 28 °, the resulting growth is used to inoculate a medium containing, in grams per liter: ammonium sulfate, 5; calcium carbonate, 9; ammoniun chloride, 1.5; magnesium ehloride.6H,O, 2; ferrous sulfate.7H20, 0.04; magnesium sulfate. 4H.,O, 0.05 ; cobalt chloride. 6H,,O, 0.001 ; zinc sulfate. 7H,_,O, 0.1; corn steep liquor, 25; starch, 55; lard oil, 2; and riboflavin, 0.002. Fermentation is carried out for 120 hr at 25 ° on a rotary shaker operating at 100 rpm. The yields of 7-ehloro-5a(lla~-dehydrotetracycline are in the range of 7-10 g/liter. The product can be isolated by partition chromatography on a Celite eolunm. The charge is prepared by acidifying the fermentation broth to pH 1.5 with cone. HCI, filtering, extracting the filtrate with n-butanol after saturating the filtrate with NaC1, and finally concentrating the butanol extract to a small volume. The Celite column is developed with a mixture of n-butanol/chloroform (80/20) saturated with 0.01 N HCI. Details of the chromatography and crystallization of the product have been described? Preparation o] Crude Enzyme Extract ]rom S. aureo]aciens AT('C 13,192. The crude extract is prepared the same as for S-adenosylmethionine :dedimethylamino - 4 - amino-anhydrotetracycline - N - methyltransferase."
D. C. Grove and W. A. Randall, "Assay Methods of Antibiotics," 238 pp. Medical Encyclopedia, New York, 1955. 5 U.S. Patent 3,007,965 (1962).
eThis volume .