Indian Journal of Experimental Biology Vol. 37, October 1999, pp. 990-994 Characterization of oxime metabolizing enzymes in a cyanobacterium Phormidium uncinatum, utilizing ethyl acetohydroximate as sole nitrogen source Suvendra N Bagchi & Thevendria M Shakila Department of Biological Sciences, R D University, Jabalpur 482001, India Received 2 December 1998; revised 10 May 1999 Ethyl acetohydroximate-adapted phototrophic cultures of Phonnidium uncinatum metabolized this oxime via hydrolysis/oxidation and dismutation of resulting hydroxylamine to nitrite and ammonia. Participating enzymes were bound to thylakoid membranes, which were solubilized and purified by conventional techniques using QAE-Sephadex and DEAE­ Cellulose columns. Specific activity of bound enzymes increased in the range of 25-75 folds. With partially purified enzymes, apparent Km values of hydrolase and oxidase for oxime and dismutase for hydroxylamine were 180, IS and 36 J.1M respectively. A number of heterotrophic nitrifying bacteria and fungi Oxime metabolism in phototrophs is hitherto can metabolize diverse N compounds as · substrates, unknown and present work elucidates the catalytic which include hydroxamates, nitroalkanes, oximes and properties of the participating enzymes after partial l hydroxylamine ,2. In some cases, free energy of purification. oxidation of these compounds has been shown to"be 3 conserved in part in A TP synthesis ,4. Enzymes like Materials and Methods pyruvic oxime oxidase, hydroxylamine dehydrogenase Axenic cultures of ethyl acetohydroximate (EAH)­ and hydroxylamine reductase have been reported, adapted strain of Phormidium uncinatum (wild type; 3 being involved in oxime metabolism . Hydroxylamine, CU 146217) was cultivated with 5 mM filter-sterilized arising from diverse sources, such as from ammonia oxime-supplemented BG-Il medium II . Checks for oxidation, is oxidized to nitrite by hydroxylamine purity, harvesting of cells and preparation of s 7 oxidase - • thylakoid membranes (particulate preparation) were 9 We have reported that upon transition from N­ carried out as described earlier , Various experiments starvation to ammonia and/or glutamine sufficiency, were done in three day old dense cultures. To check several cyanobacteria exhibit novel thylakoid bacterial contamination, culture aliquots were membrane-bound enzymatic reactions. These include, routinely transferred to nutrient broth and incubated dismutation of hydroxylamine to equimolar nitrite for 24 hr in dark. and ammonia by hydroxylamine dismutase and Cells were harvested from 5 I batches by centri­ oxidations of glutamine to hydroxylamine by fugation (5000 g). Pellets, were washed twice with glutamine oxidase and of bound hydroxylamine (bha) extraction buffer .(50 mM, Tris/HCI; pH 8.0 compounds such as oximes etc. to nitrite by bha containing 1 mM, Na-EDTA; 10 mM, MgCI2 and 8 oxidase ,9. Hydroxylamine dismutase has been 15 mM, K2HP04) and resuspended in the same buffer. JO partially purified • Cells were ruptured by sonication (15 Il amplitude for A filamentous non Nrfixing cyanobacterium, 10 min, MSE-MK Sonifier). After spinning down Phormidium uncinatum has adapted to , grow in a debries and unbroken cells at 1000 g, the crude medium containing ethyl acetohydroximate (EAH)" as extract was re-centrifuged at 4°C for 20 min at 9 sole N source . This oxime is hydrolyzed by yet 35,000 g. Negligible enzyme activitiy was detected in 9 another novel thylakoid membrane-bound enzyme, the supematant , which was discarded. The pellet called boa hydrolase. Hydroxylamine the product, is compnsmg thylakoid membrane and called dismuted to nitrite and ammonia, which are utilized particulate fraction was washed and resuspended in as N source in th~ medium. EDTA-free extraction buffer. BAGCHI & SHAKILA: OXIME METABOLIZING ENZYMES IN CY ANOBACfERIUM 991 Bound hydroxylamine (bha) hydrolase activity was Results and Discussion monitored in a reaction mixture (3 ml) containing EAH adapted strain was developed by repeated sodium pyrophosphate (PH 7.9) 150 Jlmole, MgCh; subculture of wild type P.uncinatum in the absence of 0.3 Jlmole and EAH 3 Jlmole. For hydroxylamine N03- nitrogen source, with increasing concentration 9 dismutase and bha oxidase, the reaction mixture each of oxime • The results confirm that oxime-adapted in 3 ml contained sodium pyrophosphate (PH 7.9), cultures were free from bacterial contamination. 150 Jlmole; para cresol, 0.4 Jlmole; H20 2, 200 Jlmole While both the strains grow with ammonia and and 0.9 Jlmole of the substrate-freshly prepared and exhibit activities of hydroxylamine dis mutase and bha 8 neutralized NH20H.HCI or EAH. Appropri~te oxidase , only the adapted strain could grow with amount of cyanobacterial protein (extract) was added EAH. This is due to presence of bha hydrolase in the and reactions were carried out at 30°C for 10-20 min. adapted strain which apparently is responsible for 9 Parallel controls with boiled extracts (100°C, 5 min) EAH metabolism • were also run. It has been reported earlier that hydroxylamine The reaction products N02- and NH20H were dismutase from NH/-grown wild type strain was '2 measured respectively by diazocoupling reaction solubilized by 0.8% (w/v) deoxycholate and by two­ and hydroxyquinoline method of Hewitt and step chromatography on QAE-Sephadex 25 . Yield Nicholas '3 after terminating the reaction with 5% and purification compared to particulate preparation (final concentration) of trichloroacetic acid. NH/ respectively are 19% and 30 times'o. Using the same generated was released as NH3 upon alkylation and protocol, the enzyme was purified from EAH-grown + dissolved in H2S04 as N~ by micro diffusion adapted strain (Table I). NH3 and N02- are the '4 technique • For alkylation, 40% K2C03 (final products of the reaction. In every step of purification, concentration) was used as reaction terminator production of both the products was monitored to instead of TCA. ~ + in acid was estimated exclude the possibility of the reaction being carried l5 according to Fawcett and Scott . Protein content was out by two different enzymes. The gel binding and determined by microbiuret method as has been elution properties remained unchanged. The final lO described earlier • All the assays were conducted in recovery was 26% and activity was better (46 times) triplicate and only the mean values are presented. than the wild type strain. The particulate fraction containing enzymes and Purification properties of other enzymes of EAH­ chlorophyll (6-7 mg) was suspended in solubilization adapted strain are shown in Table I. Bha oxidase and buffer (to ml) containing 25 mM, Hepes/NaOH <PH hydrolase enzymes from particulate fraction could be 8.75); 15 mM, K2HP04; 10 mM, MgCh and 0.1 mM, solubilized with deoxycholate with 90-95% original phenyl methyl sulfonyl fluoride. To this suspension, activities recovered. Oxidase was retained by sodium deoxycholate (80 mg) was added and mixture Sephadex gel and was eluted with 0.25-0.35 M salt was incubated at 4°C for 2 hr without stirring and (K2HP04). Much higher salt concentrations · (0.6- centrifuged at 57,000 g at 4°C for 120 min. 0.7 M) were required to release hydroxylamine Supernatant (8.5 ml; 1.5-2 mg mr' protein) was dismutase. Bha hydrolase also bound to above gel but appplied to a pre-equilibriated QAE-Sephadex 25 or a its activity was not recovered upon elution even with DEAE-Cellulose column (21 x2.5 cm), which was 2 M K2HP04. Either the enzyme was inactivated or first washed with 50 ml of to mM, Hepes/NaOH (PH binding was firm. This enzyme was also retained on a 8.75) containing 0.2% deoxycholate and 2 mM, DEAE-cellulose column and was eluted with 0.55-0.6 , K2HP04 before a 0-1 M, gradient of K2HP04 (PH M K2HP04• Attempt was made to use this column for 8.75) containing 0.2% deoxycholate was applied. purification of other enzymes. Activity of oxidase Active fractions (3 ml) were pooled, concentrated by was found in the washings, and of dismutase in the freeze drying, dialyzed against solubilization buffer elutes of fairly low salt concentrations (0.05-0.1 M). and chromatography step was repeated and enzymes Attempt was also made to use NaCI or respective recovered as above. substrate (1 mM) as eluents in above chromatography Fine chemicals and gel materials were bought from steps. Although some activity was recovered, the Sigma Chemical Co., St. Louis (U.S.A.) and results were lower compare to those of K2HP04, Pharmacia, Uppsala (Sweden). Other chemicals were which appeares to have some stabilizing effect (data purchased from Qualigens Fine Chemicals (India). not shown). 992 INDIAN J EXP BIOL, OCTOBER 1999 Table I-Purification profile of oxime metabolizing enzymes in EAH-adapted strain of Phorlllidiul11 IIllcillallllll Hydroxylamine dismutase (Activity is shown as N02• production and in parentheses as NH) production from NH 20 H}. Step Total activity Total protein Sp. activity Recovery Fold (11 mole min·') (mg) (11 mole min·' (%) purilll:ation mg·' protein) A 2.69 103.6 0.026 100 (2.8) (0.027) B 2.58 17.2 0.15 96 5.X (2.75) (0.16) C 1.66 5.9 0.28 62 IO. X (1.78) (0.3) o 1.4 2.1 0.66 52 25.4 (1.51 ) (0.72) E 0.7 0.58 1.2 26 46 (0.76) (1.3) Bound hydroxylamine oxidase [Units of activity is-Total = n mole min·'; and sp activity = n mole min·' mg·' protein]. Enzyme was not retai ned in DEAE-Cellulose column. A 360 120 2.6 100 I B 324 21.6 15 90 5.X 0 109 2.2 50 30 19. 2 E 82 1.05 78 22 30 Bound hydroxylamine hydrolase (Enzyme was not eluted from QAE-Sephadex column.) A 19.4 162 0.12 100 I B 19.0 19.4 0.98 98 8.2 C 12.6 1.4 9.0 65 75 Step A-particulate preparation; B-solubilized membranes; C-DEAE-cellulose chromatography; D-QAE- sephadex chromatography (1 st step); and E-QAE-Sephadex chromatography (2nd step).