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). The sample was centrifuged at 36,000 mg (dry weight), of alumina c gamma for each mg of rev/min at 20 C with an ultraviolet optical system for protein. After centrifugation, the gel was washed with recording the movement of the boundary. increasing levels of phosphate buffer (pH 7.5) to con- Sucrose-gradient centrifugation was by the proce- centrations of 0.015 M with no elution of isomerase. dure of Martin and Ames (7). The enzyme was eluted from the gel at a concentration Chemicals. Carbohydrates were purchased from the of 0.02 M phosphate in three 100-mt volumes, and this Nutritional Biochemical Corp., Cleveland, Ohio. eluate was poured directly on to a diethylaminoethyl NADH was obtained from P-L Laboratories, Milwau- (DEAE) cellulose phosphate column (25 by 5 cm). The kee, Wis. Lactic dehydrogenase and other commercial column was washed with 500 ml of 0.04 M phosphate were obtained from the Wor- buffer, and the enzyme was eluted with four 200-ml enzyme preparations volumes of 0.05 M phosphate buffer. thington Biochemical Corp., Freehold, N.J. This latter fraction was diluted to reduce the phos- RESULTS phate concentration to 1.3 x 10-2 M and allowed to flow through a small (2 by 10 cm) DEAE cellulose Purification of isomerase activity from strain phosphate column overnight. All detectable protein and 502. The initial and most extensive work on the VOL. 108, 1971 PURIFICATION OF L-FUCOSE ISOMERASE 295 purification of the enzyme was carried out with X 10-7 and V20 = 7.4 x 10- 1, a molecular the constitutive 502 strain. In this manner, large weight of 350,000 was calculated by using the quantities of enzyme could be obtained from a procedure of Schachman (15). relatively inexpensive medium. The procedure Disc-gel electrophoresis. Disc-gel electropho- used for purification of the enzyme is outlined in resis of the purified isomerase preparation also Table I and discussed in detail above. Isomeriza- indicated homogeneity. Through different experi- tion activity for L-fucose and D-arabinose was ments with various times, buffer concentrations, followed during the purification procedure, and and pH values (6.0, 7.0, and 8.5) and with pro- at no time in this or other fractionations of the tein concentrations up to 80 Mg, the purified frac- enzyme was a significant change in the ratio of tion yielded only a single band after migration. L-fucose to D-arabinose activity observed. After The purified enzyme was migrated at pH 6.0 the second ammonium sulfate precipitation, a in duplicate gels. After migration, one gel was 31-fold increase in L-fucose isomerization activity stained to identify the protein band, whereas the was obtained. Further purification techniques other gel was sliced into 1-mm thick sections such as gradient elution from DEAE cellulose which were placed in buffer and extracted to phosphate or filtration through Sephadex 200 remove as much enzyme as possible. When these yielded only a single protein peak corresponding fractions were assayed for isomerase activity, to both isomerase activities without increase in activity was found only from the region of the specific activity or change in ratio of activity sliced gel corresponding to the stained portion of (Fig. 1). One protein peak exhibiting both activi- the control gel. Similar results were obtained ties with no alteration in the ratio was observed when migration was done at pH 8.5. No change upon sucrose gradient centrifugation in the ultra- was observed in the ratio of L-fucose to D-arabi- centrifuge (Spinco, model L; Fig. 2). Sucrose nose isomerase activity in either case. From the gradient centrifugation using the method of specific activity of the purified enzyme prepara- Martin and Ames (7) indicated a sedimentation tion and densitometer scans of the gels, it was constant of 13.4S and a molecular weight of estimated that the isomerase represented about 350,000. Centrifugation in the analytical ultra- 3% of the crude extract protein. centrifuge (Spinco, model E) was performed as Figure 3 shows the results obtained after disc- previously described. gel electrophoresis of crude extracts prepared For centrifugation in the model E apparatus, a from (A) the constitutive 502 mutant grown on high molarity buffer (0.2 M potassium phosphate, casein hydrolysate, (B) the 510 constitutive mu- pH 7.0) was used to prevent aggregation of the tant grown on casein hydrolysate, (C) the wild- protein. The centrifugation was accomplished at type, PRL-R3 strain grown on casein hydroly- 36,000 rev/min and 20 C. The protein concentra- sate, (D) the wild-type, PRL-R3 strain grown on tion was adjusted to give readings of 0.423 and casein hydrolysate plus L-fucose, and (E) the 0.212 at 280 nm in the Beckman DU spectropho- purified isomerase from strain 502. Each gel re- tometer with a light path of 1 cm. The migration ceived 20 ug of protein. The band corresponding of the protein was then followed by the ultravi- in position to the purified isomerase is markedly olet optical recorder attachment on the model E increased in extracts prepared from cells induced apparatus. Only one band was recorded during (D) or constitutive (A, B) for L-fucose isomerase the forced enzyme migration at both concentra- (Fig. 3). tions of protein. Calculation of the sedimentation Activity of L-xylose. At least one of the muta- co-efficient S20,,,w (15) gave a value of 13.6S tional events required to permit growth on L-Xy- which closely approximated that obtained by the lose is a mutation for the constitutive synthesis method of Martin and Ames. Assuming D = 3 of L-fucose isomerase (5). The isomerization of TABLE 1. Purification of L-fucose isomerase from strain 502

Total activity (units) Specific activity Step (units/mg of protein) L-Fucose D-Arabinose L-Fucose D-Arabinose Crude extract ...... 31,200 23,500 1.39 0.97 Protamine sulfate supernatant fluid ...... 20,900 13,100 1.14 0.71 Ammonium sulfate precipitate ...... 24,800 16,600 4.56 2.94 Alumina c gamma gel eluate ...... 26,100 21,200 14.30 11.60 DEAEa cellulose eluate ...... 8,850 5,880 21.30 14.30 Ammonium sulfate precipitate ...... 6,300 3,640 43.50 25.10 a Diethylaminoethyl. 296 OLIVER AND MORTLOCK J. BACTERIOL.

12.0 10.0 purification, barium selectively precipitated con- E 0 taminating protein more rapidly than it activated 8.0 0 the L-fucose isomerase and could be used as a 9.6 co fractionation step. Results, however, were vari-

7.2 6.0 able and this method was not routinely incorpo- (A LU I.- iek z rated. z 4.6 4.0 m Comparison of constitutive with induced isom- 0 (A) erase. To compare the induced isomerase with a 112.0 the constitutive enzyme, the isomerase was also had I I purified from wild-type cells which been 0 20 40 60 ao 100 120 140 160 grown on a casein-hydrolysate medium supple- FRACTION NUMBER mented with L-fucose. The same purification FIG. 1. Elution profile of the purified isomerase technique was employed to yield comparable from Sephadex G-200. L-Fucose isomerase activity (0), specific activities. No differences could be ob- D-arabinose isomerase activity (U), L-xylose isom- served between the isomerase purified from ei- erase activity (0), and protein (A). A unit of enzyme ther the induced or constitutive source. The Km activity is equal to I Mmole of ketosugar formed per values for L-fucose and D-arabinose are com- min. pared in Table 2, and the reciprocal plots of ve-

E 12.0 0.5 c0 a

9 0

,3 7.2 0.3

0.2 0 .0 2.41 0.1 I,

FRACTION NUMBER FIG. 2. Sucrose density gradient centrifugation of the purified isomerase. L-Fucose isomerase activity (0), D-arabinose isomerase activity (A), and protein (0). One unit of enzyme activity is equal to 1 lmole of ke- tosugar formed per min.

L-xylose to L-xylulose was measured by using FIG. 3. Disc-gel electrophoretic patterns at pH 6.0 two different assay systems. The first involved of various crude extracts of Aerobacter aerogenes. The the incubation of L-xylose with enzyme and de- crude extracts shown were prepared from: (A) consti- termination of the L-xylulose formed by means tutive 502 mutant grown on casein hydrolysate, (B) of the cystein-carbizole test (2). The second 510 constitutive mutant grown on casein hydrolysate, assay system used a partially purified prepara- (C) wild-type PRL-R3 strain grown on casein hydroly- tion of L-xylulokinase to couple L-xylulose phos- sate, (D) wild-type PRL-R3 strain grown on casein phorylation through the spectrophotometric hydrolysate plus L-fucose, and (E) purified isomerase from strain 502. assay for kinase activity (3). In both cases, double reciprocal plots of velocity versus sub- TABLE 2. Comparison ofpurified isomerase from strate concentration yielded an apparent Km strains PRL-R3, 502, and 531 value of 0.4 M for L-xylOse, although L-xylose actually became inhibitory at concentrations over Purifi- Ratio of Strain cation Substrate 0.25 M. The extrapolated Vma,, for L-xylose only Km. (M) activity (fold) at Vmax represented 0.83% of the value for L-fucose or D- arabinose. PRL-R3 31 L-FucOse 0.055 1.0 Effect of metals. As previously reported (2), D-Arabinose 0.140 1.0 manganous ion was required for activity. Other divalent metallic ions such as magnesium, cobalt, 502 29 L-Fucose 0.050 1.0 and ferrous could partially replace the man- D-Arabinose 0.160 1.0 ganous requirement. Barium, mercury, cuprous, 531 40 L-Fucose 0.035 1.0 and nickel ions inactivated the isomerase com- ID-Arabinose 0.098 0.45 pletely. After the first ammonium sulfate step in VOL. 108, 1971 PURIFICATION OF L-FUCOSE ISOMERASE 297 locity versus substrate concentration are com- pared in Fig. 4. A similar Vmax was obtained for both D-arabinose and L-fucose. As previously reported, dithiothreitol was a competitive inhib- itor for either substrate (13). This compound was also effective in protecting the isomerase against any noticeable heat inactivation when subjected to 55 C for 10 min (Fig. 5) and al- lowed only a 20% loss of activity after 25 min. Both substrate activities were equally protected by the presence of dithiothreitol; however, in the absence of this compound, they declined in paral- lel, losing over 50% of their activity in the first 10 min and over 80% in 25 min. When L-fucose was present in sufficient quantity to give max- imal activity, the addition of D-arabinose did not result in an increase in activity. Mutant strain 531. In one purification experi- ment, a difference in the normal L-fucose to D- TIME (min., 55C) arabinose ratio was observed. With the activity FIG. 5. Inactivation of the isomerase at 55 C in the normal substrate concentrations of 6.7 x 10-2 M presence and absence ofdithiothreitol (DTT) (1O-3 M). The plot of velocity over initial velocity (v/v0) shows a strong protective effect of DTT. Since the activities of L-fucose and D-arabinose decline in parallel and the ratio remains constant, the curves for both sugars su- 2.0 00 perimpose.

used in the assay system, the D-arabinose activity 1.0 was now greater than the L-fucOse activity; this new ratio of activity was maintained during the entire fractionation procedure. The D-arabinose- grown culture of strain 502 which had been used as inoculum for this particular experiment was examined and found to consist of two types of 4.0 cells. One type showed the normal isomerase ac- tivity, whereas the second possessed an altered D- arabinose to L-fucose activity ratio. The double C) 2.0 reciprocal plot of velocity versus substrate con- 0-a wi centration (molarity) of the isomerase purified (40-fold) from this new strain are shown in Fig. 4. Strain 531 appears to produce an altered isomerase with an apparent decrease in the Km value for D-arabinose and perhaps L-fucose Table 2). Organisms possessing the altered isomerase ratio could be selected from the original popula- tion simply by plating the constitutive mutant on D-arabinose and picking the large colony types. Further screening showed a one-to-one corre- spondence of colony size to the new ratio of L- fucose to D-arabinose. The large colonies once 1/SUBSTRATE M picked and grown on D-arabinose or casein hy- FIG. 4. Comparison of the double reciprocal plots drolysate always produced the novel ratio. The of velocity versus substrate concentration for the L- small colonies, on the other hand, always pro- fucose isomerase from three strains of Aerobacter aer- duced organisms, the isomerase activity of which ogenes: top, wild-type PRL-R3 strain; center, 502 con- stitutive mutant; and lower, 531 constitutive mutant. L- was greater on L-fucOse than D-arabinose. Fucose (0), D-arabinose (0). Velocity is in terms of Mutant strain 534. Strain 534 was obtained micromoles per minute. from strain 531; strain 534 possessed a greatly 298 OLIVER AND MORTLOCK J. BACTERIOL. decreased growth rate on D-arabinose (from 0.66 stitutive synthesis of enzymes of the L-fucose generations per hour for strain 531 to 0.12 gener- catabolic pathway. The isomerization of D-arab- ations per hour for strain 534) but had a normal inose to D-ribulose is catalyzed by an L-fucose growth rate for L-fucose. The regulation of isom- isomerase which appears to be unaltered in its erase activity appears to be at least partially re- normal catalytic activity or physical properties. stored in this mutant and is induced by the pres- This isomerase was purified to apparent homoge- ence of L-fucose in the medium. Table 3 gives the neity from an inducible wild-type PRL-R3 isomerase activities of crude extracts of the wild- strain and two constitutive mutants. Homoge- type PRL-R3 strain, and strains 502, 531, and neity was indicated by a single protein peak ex- 534. The concept of an altered isomerase in hibiting both isomerase activities, by using the strain 531 is supported by the induction of a sim- following procedures: (i) disc-gel electrophoresis, ilar substrate activity ratio in strain 534 by L- (ii) ultracentrifugation in both the model E and fucose. model L ultracentrifuge, (iii) Sephadex gel filtra- tion, and (iv) ion-exchange chromatography. The apparent Km of the enzyme from the wild DISCUSSION type and 502 constitutive mutant was the same A. aerogenes possesses inducible enzyme path- for both L-fucOse and D-arabinose (0.15 and 0.05 ways to permit it to degrade three of the aldo- M, respectively). The maximal velocity catalyzed pentoses (D-ribose, D-xylOse, L-arabinose) and by the enzyme from either organism was iden- two of the pentitols (ribitol, D-arabitol). Mutants tical for both sugars. At maximal catalytic ve- can be selected for growth on many of the less locity for L-fucose, the addition of D-arabinose common pentoses and pentitols, including D- did not result in increased activity. The Vma. arabinose, D-lyxOse, L-xylose, L-lyxose, xylitol, data would appear to indicate indirectly that the and L-arabitol. In each case, the mutant pos- sugars are acted on at the same catalytic site. sesses the necessary enzyme activity to convert Dithiothreitol competitively inhibited activity the new substrate to a structure which is interme- for both substrates, exhibiting an apparent K, diate in the pathway of degradation of a natural which was equal for either substrate (13). This substrate (11). Growth on xylitol and L-arabitol compound protected activity on both sugars is made possible by a mutation to the constitu- against noticeable heat inactivation at 55 C. tive synthesis of ribitol dehydrogenase. The con- Both activities were shown to decline in parallel stitutively synthesized dehydrogenase then cata- at 55 C, however, in the absence of this agent. lyzes the oxidation of xylitol to D-xylulose or L- The inhibition data would also tend to support arabitol to L-xylulose (10). The mutation permit- the thesis that the sugars are binding at the same ting growth on xylitol has recently been mapped site. as adjacent to the ribitol dehydrogenase struc- Activity was stimulated by the addition of the tural gene (W. T. Charnetzky and R. P. Mort- same divalent metallic ions. No protective effect lock, Bacteriol. Proc., p. 137, 1970). Wu et al. was found, however, during heat inactivation due have described additional mutations which to presence of divalent ions. permit faster growth with xylitol as the substrate. Continued selection on D-arabinose has re- Among such mutations is an apparent alteration sulted in the isolation of an additional mutant in the structural gene for ribitol dehydrogenase, (strain 531) which has an alteration in the ap- resulting in a change in Km and Vmax values for parent Km of the enzyme for D-arabinose isomer- xylitol (I 7). ization. This alteration in ratio can be directly Growth on D-arabinose also results from a correlated to colony size of the organism when regulatory mutation, one which permits the con- grown on D-arabinose. TABLE 3. D-Arabinose isomerase activity ofcrude extracts and ratio of D-arabinose to L-fucose activity Growth substrate

Strain Casein hydrolysate D-Arabinose L-Fucose Units/mg of Ratio Units/mg of Ratio Units/mg of Ratio protein protein protein PRL-R3 0.01 0.01 1.40 0.60 502 0.97 0.70 0.58 0.60 1.76 0.62 531 4.70 1.40 1.50 1.34 1.46 1.39 534 0.19 1.30 0.Ola 4.60 1.60 a Incubation with D-arabinose for 6 hr. Substrate concentration for assay was 0.067 M. VOL. 108, 1971 PURIFICATION OF L-FUCOSE ISOMERASE 299

L-Fucose isomerase also catalyzes the isomeri- 4. Bisson, T. M., E. J. Oliver, and R. P. Mortlock. 1968. zation of L-xylose to L-xylulose, and mutants Regulation of pentitol metabolism by Aerobacter aerog- enes. II. Induction of the ribitol pathway. J. Bacteriol. which are capable of growth on L-xylose are con- 95:932-936. stitutive for this enzyme. Although mutants ca- 5. Camyre, K. P., and R. P. Mortlock. 1965. Growth of pable of growth on D-arabinose can be selected Aerobacter aerogenes on D-arabionse and L-xylose. J. from a&population of 107 cells in 2 to 3 days, 40 Bacteriol. 90:1157-1158. 6. Colowick, S. P. 1955. Separation of proteins by use of to 60 days are to mutants required select for adsorbents, p. 90-98. In S. P. Colowick and N. 0. Ka- growth on L-xylose from a similar population of plan (ed.), Methods in enzymology, vol. 1. Academic cells (11). It is possible that for L-xylose an addi- Press Inc., New York. tional mutation is required for growth, perhaps 7. Martin, R. G., and B. N. Ames. 1970. A method for deter- mining the sedimentation behavior of enzymes: applica- facilitiating the entrance of L-xylOse into the cell. tion to protein mixtures. J. Biol. Chem. 236:1372-1379. Studies of the mutation permitting growth on 8. Mortlock, R. P. 1966. D-Arabinose isomerase, p. 583-585. L-lyxose have been hampered by the lack of suf- In W. A. Wood (ed.), Methods in enzymology, vol. 9. ficient quantity of pure L-lyxose. Anderson and Academic Press Inc., New York. 9. Mortlock, R. P., D. D. Fossit, D. H. Petering, and W. A. Wood (2) detected both L-lyxose and L-xylose Wood. 1965. Metabolism of pentoses and pentitols by isomerization activity in cell-free extracts pre- Aerobacter aerogenes. III. Physical and immunological pared from cells grown on L-xylose. By using a properties of pentitol dehydrogenases and pentuloki- commercial preparation for L-lyxose, we nases. J. Bacteriol. 89:129-135. found 10. Mortlock, R. P., D. D. Fossit, and W. A. Wood. 1965. A mutants selected for growth to be constitutive for basis for the utilization of unnatural pentoses and penti- L-fucose isomerase; however, with the same prep- tols by Aerobacter aerogenes. Proc. Nat. Acad. Sci. aration, we have not been able to detect isomeri- U.S.A. 54:572-579. zation activity by using the purified L-fucose 11. Mortlock, R. P., and W. A. Wood. 1964. Metabolism of pentoses and pentitols by Aerobacter aerogenes. I. isomerase. Demonstration of pentose isomerase, pentulokinase, and pentitol dehydrogenase enzyme families. J. Bacteriol. 88: 828-834. ACKNOWLEDGMENTS 12. Oliver, E. J., T. M. Bisson, D. J. LeBlanc, and R. P. Mort- lock. 1969. D-Ribulose production by a mutant of Aero- This work was supported by Public Health Service research bacter aerogenes. Anal. Biochem. 27:300-305. grant AI-06848 from the National Institute of Allergy and 13. Oliver, E. J., and R. P. Mortlock. 1969. Competitive inhi- Infectious Diseases and by grant Gb-3864 from the National bition of an L-fucose isomerase activity by dithiothreitol. Science Foundation. Biochem. Biophys. Res. Commun. 36:24-29. 14. Oliver, E. J., and R. P. Mortlock. 1971. Growth of Aero- bacter aerogenes on D-arabinose: origin of the enzyme LITERATURE CITED activities. J. Bacteriol. 108:287-292. 1. Adelberg, E. A., M. Mandel, and G. C. C. Chen. 1965. 15. Schachman, H. K. 1957. Ultracentrifugation, diffusion and Optimal conditions for mutagenesis by N-methyl-N'- viscometry, p. 32-104. In S. P. Colowick and N. 0. nitro-N-nitrosoguanidine in Escherichia coli K-12. Kaplan (ed.), Methods in enzymology, vol. 9, Academic Biochem. Biophys. Res. Commun. 18:788-795. Press Inc., New York. 2. Anderson, R. L., and W. A. Wood. 1962. Pathways of L- 16. Warburg, O., and W. Christian. 1941. Isolierung und xylose and i-lyxose degradation in Aerobacter aerog- Krystallization des Garungsferments. Enolase. Biochem. enes. J. Biol. Chem. 237:296-303. Z. 310:384-421. 3. Anderson, R. L., and W. A. Wood. 1962. Purification and 17. Wu, T. T., E. C. C. Lin, and S. Tanaka. 1968. Mutants of properties of L-xylulokinase. J. Biol. Chem. 237:1029- Aerobacter aerogenes capable of using xylitol as a novel 1033. carbon source. J. Bacteriol. 96:447-456.