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Metabolism of D-Arabinose by Aerobacter Aerogenes: Purification of the Isomerasea

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Metabolism of D-Arabinose by Aerobacter Aerogenes: Purification of the Isomerasea JOURNAL OF BACrERIOLOGY, Oct. 1971, p. 293-299 Vol. 108, No. I Copyright 0 1971 American Society for Microbiology Printed in U.S.A. Metabolism of D-Arabinose by Aerobacter aerogenes: Purification of the Isomerasea EUGENE J. OLIVER2 AND ROBERT P. MORTLOCK Department of Microbiology, University of Massachusetts, Amherst, Massachusetts 01002 Received for publication 5 April 1971 In Aerobacter aerogenes, the mutational event permitting the utilization of D- arabinose as a source of carbon and energy is a regulatory mutation resulting in the constitutive synthesis of certain enzymes of the L-fucose catabolic pathway. L- Fucose isomerase catalyzes the isomerization of D-arabinose to D-ribulose. This enzyme was purified to homogeneity as indicated by a single band in disc-gel elec- trophoretic columns and single peaks with column chromatography and ultracen- trifugation from the wild-type PRL-R3 strain, induced with L-fucose and two con- stitutive mutants, 502 and 510. The ratios of the activities of this isomerase on D- arabinose and L-fucose remained constant throughout all purifications. The ap- parent Km of the isomerase from the wild-type strain induced with L-fucose and from the constitutive mutant strains was 5.0 x 10-2 M for L-fucose and 1.5 x 10-1 M for D-arabinose. A strain 531 possessing an apparent alteration in the isomerase was isolated from the strain 502. This altered isomerase exhibited a lowered Km for D-arabinose. In previous papers, evidence has been pre- enes PRL-R3. Strain 502 was constitutive for L-fucose sented that the initial mutational event permit- isomerase activity and was isolated from PRL-R3 by ting the growth of Aerobacter aerogenes strain selection on D-arabinose-salts medium. Strain 531 was PRL-R3 on D-arabinose is a regulatory muta- isolated from a culture of 502 which had been main- tion. This mutation permits the constitutive syn- tained by transfer on slants of D-arabinose-salts agar of of the L-fucose catabolic for several years. Strain 534 was isolated from 531 for thesis enzymes its lack of ability to utilize D-arabinose after treatment pathway. L-Fucose isomerase (EC 5.3.1.3) cata- with N-methyl-N'-nitro-N-nitrosoguanidine and peni- lyzes the isomerization of D-arabinose to D-ribu- cillin enrichment (1, 4). lose (5, 15). The latter ketopentose is a normal The basic growth medium consisted of minimal salts intermediate in ribitol degradation and induces (12) supplemented with 0.5% of carbohydrate or 1% both enzymes of the ribitol catabolic pathway: casein hydrolysate (vitamin-free, salt-free, acid hydro- ribitol dehydrogenase (EC 1.1.1.56) and D-ribu- lyzed, Nutritional Biochemicals, Inc.). All cultures lokinase (EC 2.7.1.47). The induced kinase then were grown aerobically at 30 C. For purposes of en- catalyzes the second step for the degradation of zyme purification, cells were grown in New Brunswick D-arabinose (4). A partial purification of this microferm fermentors. The 12-liter fermentation tanks contained 10 liters of medium and were sparged with isomerase has been described (5, 8). air at 4.5 liters per minute and agitated by a rotor This paper described the further purification turned at 250 rev/min. Growth was measured as de- and some of the properties of L-fucose isomerase scribed previously (14). and an apparent mutant form of the enzyme Determination of enzyme activity. Assays for isomer- which possesses increased activity for D-arabi- ization of D-arabinose and L-fucose were performed nose as a substrate. spectrophotometrically by observing the rate of reduced nicotinamide adenine dinucleotide (NADH) oxidation MATERIALS AND METHODS at 340 nm with a multiple absorbance spectropho- Bacterial strains, media, and growth conditions. The tometer (Gilford, model 2000). The assay was per- parent strain used in this investigation was A. aerog- formed in microcuvettes, each cuvette containing 33 nmoles of MnCI2; 1.25 umoles of tris(hydroxymethyl)- aminomethane (Tris)-hydrochloride buffer, pH 7.0; 1 ' This paper was presented in part at the 68th Annual Meeting of the American Society for Microbiology, Detroit, Mmole of reduced glutathione; 10 gmoles of substrate; Mich., 5-10 May 1968. 50 nmoles of NADH; excess purified ribitol dehydro- 2 Present address: Department of Biochemistry, Division of genase and isomerase in a total volume of 0.15 ml. The Enzymology, National Institutes of Health, Bethesda, Md. isomerization of L-xylose to L-xylulose was measured 20014 from the rate of pentulose formation by the method of 293 294 OLIVER AND MORTLOCK J. BACTERIOL. Anderson and Wood (2) and also spectrophotomet- isomerase activity was retained by this column. The rically. The latter assay system contained L-Xylose enzyme was then eluted from this small column by and isomerase; excess purified L-xylulokinase (EC washing with two 10-ml volumes of high concentration 2.7.1.d); 0.5 A.tmoles of adenosine triphosphate (ATP); phosphate buffer (0.2 M, pH 7.5). A second ammonium 1.0 Mmole of MgCl2; 40 nmoles of MnCI2; 1.5 nmoles sulfate precipitation was employed at this point by the of reduced glutathione; 8 Amoles of Tris-hydrochloride addition of a saturated (4 C), neutralized (pH 7.0) so- buffer (pH 7.0); 250 nmoles of phosphoenolpyruvate; lution of ammonium sulfate. The fraction precipitating 50 nmoles of NADH; and lactic dehydrogenase con- between 37 and 45% saturation contained the highest taining pyruvate kinase (Worthington Biochemical isomerase activity. This was the fraction used in the Corp.) in a total volume of 0.15 ml. One unit of isomer- studies presented in this paper. ase was that amount of enzyme which catalyzed the Analytical techniques. Polyacrylamide gel electro- formation of 1 Mmole of product per min. Specific ac- phoresis was carried out in a disc electrophoresis appa- tivity is expressed as units per milligram of protein. ratus (Canalco, model 6) with an electrophoresis power Protein determination was made by the method of supply (Arthur H. Thomas, Philadelphia, Pa.) used to Warburg and Christian (16). Dilutions made for the maintain constant amperage. The acrylamide gels were purpose of isomerase assay were made in 10-3 M phos- prepared by using Cyanogum 41 (Fisher Scientific Co., phate buffer containing 5 x 10-4 M MnCl2 and 5 x 10-3 Fairlawn, N.J.) as the gelling agent at a concentration M reduced glutathione at pH 7.5. of 7.5% (w/v) in 24 ml of buffer mixed with 0.06 -ml of Purification of enzymes. Ribitol dehydrogenase and N, N, N', N'-tetramethylethylenediamine (TEM ED) as L-xylulokinase were purified as described previously accelerator and 0.6 ml of 7% (w/v) ammonium persul- (10, 11). fate as catalyst. The gels were polymerized at room For the purification of L-fucose isomerase, 10 to 20 temperature to form gel columns (5 by 50 mm). liters of cells were harvested in the late exponential Electrophoretic migration of the proteins was carried growth phase with a Sharples steam-driven centrifuge. out in 0.2 M glycine-hydrochloride (pH 6.0) or Tris- Cells were washed with distilled water and resuspended hydrochloride buffer at pH 7.0 or 8.5, respectively. in 10-3 M phosphate buffer containing 5 x 10-4 M Samples were prepared at a concentration of 1.0 ethylenediaminetetraacetic acid (EDTA), 5 x 10-4 M mg/ml in a 20% sucrose solution for application to the MnCl2, and 10-S M mercaptoethanol at pH 7.5. The top of the gels and applied in 2- to 80-Mliter volumes. buffer referred to throughout this purification proce- The electrophoresis was always accomplished at 4 C, dure was of similar composition and pH with the ex- using precooled buffer systems, with a current of 8 ception of the phosphate concentrations. The tempera- ma/tub for a duration of from 3 to 6 hr. After the gels ture was maintained at 4 C throughout the purification were removed, they were stained with 1% amido black procedure. Cells were broken by means of the Ribi (in 7% acetic acid) for 30 min. The gels were destained refrigerated cell fractionator (Ivan Sorvall, Inc.). electrophoretically with 7% acetic acid as the con- ducting fluid. The gels were scanned by using a re- The first two fractionation steps are similar to those cording densitometer (Photovolt) or were recorded previously described for a partial purification of the photographically with high-contrast copy film (Kodak, enzyme (8). After centrifugation at 31,000 x g to re- HC 135-36). move cell debris, the supernatant fluid was diluted to To recover the enzyme activity, the gels were sliced adjust the protein concentration to 10 mg/ml. Ammo- with a razor blade into 1-mm thick wafers and placed nium sulfate was added to 0.1 M, and a solution of pro- in 0.5 ml of buffer for extraction. tamine sulfate (18 mg/ml) was added to give a final Ultracentrifugation was performed in the analytical protamine concentration of 1.8 mg/ml. After centrifu- ultracentrifuge (model E; Beckman Instruments, Inc., gation, ammonium sulfate was added to the superna- Spinco Division, Palo Alto, Calif.) fitted for recording tant fluid, and the protein precipitating between 40 and with the type RS Dynograph attachment (Beckman 60% saturation was collected by centrifugation. This Instruments Inc., Offner Division, Schulter Park, Ill.). fraction was dissolved and diluted in buffer to lower The protein sample used for analysis was put in a 12- the ammonium sulfate concentration to 0.02 M. After mm, Epon-filled, double-sectored (2.5 inch) cell, with dilution, the protein concentration was 1 mg/ml. A sapphire windows, and run in an An-H Titanium rotor suspension of alumina c gamma was added to give 1.5 (Beckman).
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