JOURNAL OF BACTERIOLOGY, Oct. 1985, p. 479-483 Vol. 164, NO. 1 0021-9193/85/090479-05$02.00/0 Copyright C 1985, American Society for Microbiology Inactivation of Glycerol Dehydrogenase of Klebsiella pneumoniae and the Role of Divalent Cations E. A. JOHNSON,1 R. L. LEVINE,2 AND E. C. C. LIN'* Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 021151 and Laboratory ofBiochemistry, National Heart, Lung, and Blood Institute, Bethesda, Maryland 202052 Received 17 December 1984/Accepted 24 June 1985 Anaerobically induced NAD-linked glycerol dehydrogenase of Klebsiella pneumoniae for fermentative glycerol utilization was reported previously to be inactivated in the cell during oxidative metabolism. In vitro inactivation was observed in this study by incubating the purified enzyme in the presence of 02, Fe2+, and ascorbate or dihydroxyfumarate. It appears that 02 and the reducing agent formed H202 and that H202 reacted with Fe2+ to generate an activated species of oxygen which attacked the enzyme. The in vitro-oxidized enzyme, like the in vivo-inactivated enzyme, showed an increased Km for NAD ( but not glycerol) and could no longer be activated by Mn2+ which increased the Vk of the native enzyme but decreased its apparent affinity for NAD. Ethanol dehydrogenase and 1,3-propanediol oxidoreductase, two enzymes with anaerobic function, also lost activity when the cells were incubated aerobically with glucose. However, glucose 6-phosphate dehydrogenase (NADP-linked), isocitrate dehydrogenase, and malate dehydrogenase, expected to function both aerobically and anaerobically, were not inactivated. Thus, oxidative modification of proteins in vivo might provide a mechanism for regulating the activities of some anaerobic enzymes.

Glycerol dehydrogenase (glycerol:NAD+ 2-oxidoreduc- integrity, as indicated by its capacity to bind to specific tase) catalyzes the first step in the fermentative utilization of antibodies and its close copurification with the active en- glycerol by Klebsiella pneumoniae. The product, zyme. However, a local structural change at the active site is dihydroxyacetone (DHA), is phosphorylated by an ATP- betrayed by the loss of apparent affinity for NAD (31). dependent kinase. The NAD consumed is in part regener- In vitro oxidative inactivation was observed in glutamine ated in a two-step parallel pathway in which glycerol is synthetase (18) and a number of other enzymes from procar- converted by a B12-dependent dehydratase to 3- yotic or eucaryotic origins (7). A mixed-function oxidation hydroxypropionaldehyde, which is then reduced to 1,3- system (MFO) seems to be implicated as follows: propanediol by an NADH-dependent enzyme. Glycerol dehyrogenase, DHA kinase, glycerol dehydratase, and 1,3- NAD(P)H + H+ + 02 MF9 H202 + NAD(P)+ propanediol oxidoreductase are members of the dha regulon NAD(P)H + 2Fe3+ MF9 2Fe2+ + NAD(P)+ + H+ under repressor control (5, 13, 20, 24, 29). Exposure to 02 Of cells growing anaerobically on glycerol results in irreversible Fe2+ + H202-- activated oxygen + Fe3+ inactivation of glycerol dehydrogenase with no effect on Activated + -* oxidized DHA kinase (20, 30, 31). The loss of glycerol dehydrogenase oxygen enzyme enzyme activity can have a further effect in curtailing the expression NAD(P)H may be replaced by a variety of other reducing of the entire dha system by cutting the supply of DHA, the agents, depending on the MFO system. The involvement of inducer (4). Even in a repressor-constitutive strain, which oxidative biochemical reactions in this process and the does not require an inducer, 02 can still repress at least DHA interesting fact that most of the target enzymes possess a kinase synthesis (30). In any event, with the appearance of nucleotide-binding site and depend on a divalent metal 02 the flow of glycerol is diverted through the aerobically cation for catalytic activity (7) raised the possibility that induced glp system in which glycerol is first phosphorylated glycerol dehydrogenase is inactivated in a similar manner. by an ATP-dependent kinase. The product, sn-glycerol Effect of divalent metal ions. Glycerol dehydrogenase was 3-phosphate, is then dehydrogenated to DHA phosphate by purified from K. pneumoniae ECL2106, a dha constitutive a flavoprotein linked to an electron transfer chain (19). mutant of strain ECL2103 (30, 31). Cells from 2-liter Glycerol dehydrogenase, functional as a dimer or a anaerobic cultures (36) were collected by centrifugation, tetramer of a 40-kilodalton peptide, is activated by K+ or washed with 50 mM sodium-potassium phosphate (pH 7.0), NH4'. The latter decreases the Km for glycerol more than suspended in the same buffer, and sonicated while being 20-fold but does not affect the Km for NAD (21, 22). Even chilled (5). The extract was cleared by centrifugation at when an activating monovalent cation is present in excess, 20,000 x g for 20 min (4°C). Solid ammonium sulfate was the enzyme activity can be further enhanced by Mn2+ (12). mixed with the extract to 55% saturation. The In contrast, irreversible inactivation of the enzyme occurring slowly in vivo requires aerobic metabolism of a carbon source (20). pelleted protein was dissolved in 20 mM Tris hydrochloride inactivated enzyme retains its general structural (pH 8.0) and dialyzed against the same buffer for 12 h at 4°C. Freshly The dialyzed extract (2 ml) was loaded onto a column for anion-exchange high-pressure liquid chromatography (13) * Corresponding author. and eluted with 35 ml of 20 mM Tris hydrochloride (pH 8.0) 479 480 NOTES J. BACTERIOL.

ing on the particular protein preparation. Fe2" had no significant effect even at 50 ,uM. Zn2+ at 50 ,uM was inhibitory, as previously reported (21). The effect of Mn2+ (from 0.2 to 2 ,uM) was greater in the presence of twice the concentration of o-phenanthroline. This was probably be- cause the excess chelators removed an inhibitory cation from the enzyme. A slight activation of a purified prepara- tion of the enzyme by this chelator in the absence of Mn2+ was previously reported (22). Activation by Mn2+ was rapid (full effect attained in <30 s) and reversible (passage of the Mn2+-treated enzyme through a Sephadex G-25 column, which took 15 min, reduced its activity to the basal level). Glycerol dehydrogenase not treated with Mn2+ did not lose activity by this desalting procedure. Mn2+ activated the enzyme by increasing the Vmax at the expense of diminishing the apparent affinity of the enzyme for NAD+. In the X; X absence of added Mn2 , the Km for NAD+ was about 64 ,uM; the addition of 25 ,uM Mn2+ and 50 jiM o-phenanthroline &4 -\ increased the value to 230 ,uM. In agreement with an earlier observation made with the enzyme in a cell extract (12), the Km of the purified enzyme for glycerol (about 3.5 mM) was not changed by the addition of Mn2+. 2 - Results from this and previous work indicate that there are two different divalent cation-binding sites on the enzyme. One site binds the metal ion so tightly that no significant dissociation occurs during passage of the protein through a 0 1 2 column. Under certain conditions, however, this metal ion HOURS can be removed or blocked by specific chelators, resulting in FIG. 1. Inactivation of glycerol dehydrogenase (GDH) by Fe and total inactivation of the enzyme. The other site loosely binds ascorbate and protection by catalase and superoxide dismutase. Mn2+. The steady-state rates of catalysis for several pyridine Purified glycerol dehydrogenase (3.5 nM) was incubated undis- nucleotide-linked dehydrogenases are known to be limited turbed in borosilicate tubes (13 mm diameter) containing 0.1 ml of 50 by the dissociation of NADH from the enzyme (27). The mM HEPES (pH 7.3) and 25 ,uM FeCl3 with no other addition (0), with 25 mM ascorbate (0), with 25 mM ascorbate and 125 U of enhancement of the enzyme activity by Mn2+ might be the superoxide dismutase (1 ,uM) (A), or with 25 mM ascorbate and 186 result of stabilizing the conformation that allows ready U of catalase (1 FLM) (A). NADH dissociation. In vitro enzyme inactivation. A simple chemical system consisting of molecular oxygen, a reducing agent such as with a 0 to 0.5 M NaCl gradient. Active fractions were ascorbate, and iron was found to mimic the effect of an MFO pooled and applied to a column of Ultrogel ACA-34 and on a target protein by generating a species of activated eluted with 50 mM phosphate buffer. In both of these steps oxygen (7). When this test (17) was applied to glycerol the enzyme eluted as a single peak. The partially purified dehydrogenase, it was found that aerobic incubation of the dehydrogenase was loaded onto a blue-Sepharose CL-6B enzyme with iron and ascorbate resulted in a 75% loss of column (1 by 6 cm), washed with 30 ml of phosphate buffer, activity in 2 h. The addition of catalase significantly retarded and eluted in the same buffer supplemented with 2 mM this loss. The addition of superoxide dismutase (the copper- NAD. The enzyme eluted as two peaks of activity during this and zinc-containing bovine enzyme) also protected the pro- last step; the first (3% recovery) did not bind to the gel, tein, although the effect was less striking. No inactivation whereas the second (16% recovery) required NAD for its occurred when the enzyme was incubated with iron in the elution. The major fraction was used. Sodium dodecyl absence of a reducing agent (Fig. 1). The rate of inactivation sulfate-polyacrylamide gel electrophoresis of the final prep- of glycerol dehydrogenase by 02 and 25 mM ascorbate aration showed a single protein band with a molecular weight increased fivefold as the amount of FeCl3 was varied from 1 of 44,000 3,000 (15). The elution of the enzyme from a to 25 ,uM. No inactivation occurred in the presence of column of calibrated ACA-34 Ultrogel indicated that the ascorbate when iron was replaced by an equal molar con- enzyme was active predominantly as a tetramer. Enzyme centration of Mn2+ (25 jiM). Similar results (73% loss of activity was measured spectrophotometrically by the initial activity in 2 h) were obtained when 1 mM dihydroxyfumar- rate of NADH formation caused by glycerol. The assay ate was used as the reducing agent in the presence of 10 ,uM mixture contafined 100 mM glycerol, 0.6 mM NAD, 30 mM Fe2 ammonium sulfate, and 100 mM potassium bicarbonate at Reactivation of the modified enzyme. Glycerol dehydroge- pH 9.0 (29). Protein concentration was determined with the nase inactivated by ascorbate and iron was not revived by Coomassie blue reagent (2). Specific activities were ex- passage of the protein through a Sephadex G-25 column. Nor pressed as micromoles per minute per milligram of protein at was the activity restored by treatment with 10 mM dithio- 300C. threitol. The enzyme was no longer stimulated by Mn2+. In contrast to partially purified glycerol dehydrogenase However, when the concentration of NAD was raised from (21, 22), the enzymre purified to electrophoretic homogeneity 0.6 to 10 mM in the assay mixture the enzyme activity was was completely inhibited by 1 mM o-phenanthroline or about 80% restored (Table 1). A similar reactivation was EDTA. In the absence of these chelators, the enzyme observed with the enzyme inactivated in vivo (31). Perhaps activity was stimulated 2- to 13-fold by 1 ,uM Mn2", depend- not by coincidence, when in vivo-inactivated glycerol dehy- VOL. 163, 1985 NOTES 481 drogenase was tested with Mn2+, no stimulation of activity TABLE 2. Effect of aerobic incubation of cells on the activity of was observed. Thus, the in vivo- and in vitro-modified pyridine nucleotide-linked dehydrogenases enzymes resembled each other in two different kinetic char- Activity (,umol acteristics. min-' mg of pro- tein-') at 30°C Effects of metal ions on in vivo inactivation. To see whether Enzyme the external presence of divalent cations could affect the in After 4 h Inactivation Zero of aerobic vivo inactivation process, cells grown anaerobically on time incubation glycerol were transferred to mineral medium (36) with chlor- amphenicol (10 ,ug/ml) and incubated aerobically for 2 h Glycerol dehydrogenase 0.75 0.12 84 under different conditions. The specific activity remained the 1,3-Propanediol oxidoreductase 0.30 0.09 70 same if no further additions to the medium were made. The Ethanol dehydrogenase 0.25 0.07 72 addition of glucose (20 mM) alone resulted in a 44% loss of Glucose 6-phosphate dehydroge- 0.15 0.15 0 activity. The addition of Mn2+ (100 ,uM) with the glucose nase prevented the loss. The addition of Fe2+ (100 ,uM) with the Isocitrate dehydrogenase 0.04 0.05 0 glucose increased the loss to 67%. Similar results were found Malate dehydrogenase 0.88 0.92 0 whether or not assays of activity were done in the presence of 25 p.M Mn2+ and 50 ,uM o-phenanthroline. It thus seems that the divalent cation composition in the incubation me- dium influenced the intracellular environment and that the and malate dehydrogenase activities were assayed as de- iron-mediated oxidative modification of glycerol dehydroge- scribed in the Worthington Diagnostics enzyme manual of nase also occurred in vivo. 1978. Isocitrate dehydrogenase activity was assayed by the In vivo inactivation of other anaerobic dehydrogenases. method of Cleland et al. (3). 1,3-Propanediol oxidoreductase and ethanol dehydrogenase, After cells grown anaerobically on glycerol were incu- like glycerol dehydrogenase, become gratuitous under aero- bated aerobically for 4 h in a glucose medium without a bic conditions. Therefore, these enzymes were also tested nitrogen or sulfur source, the activities of the anaerobic for aerobic inactivation in vivo. Glucose 6-phosphate enzymes were largely lost, but the activities of the control dehyrogenase (NADP-linked), isocitrate dehydrogenase, enzymes were unchanged (Table 2). and malate dehydrogenase, which should be functional both Possible mechanism for respiratory control of enzyme ac- aerobically and anaerobically, were used as controls. tivity. Oxidative inactivation of key metabolic enzymes in K. pneumoniae ECL2103, lacking alkaline phosphatase vitro was shown previously to affect covalent bonds (7). By but wild type in the dha system, was grown anaerobically in the model proposed, Fe2+ first binds to a specific site on the 6 liters of medium (36) containing 20 mM glycerol. When the target protein; the hydrogen peroxide generated by aerobic culture reached 80 U (Klett-Summerson colorimeter with a metabolism then oxidizes the enzyme-bound Fe2+, yielding no. 42 filter), the cells were harvested by centrifugation, Fe3+ and a radical that has a high probability of attacking the washed in 50 mM of mineral medium (pH 7.0), and sus- nearest reactive amino acid side chain. In the case of pended in 2 liters of the mineral medium supplemented with glutamine synthetase, one particular histidine residue is 20 mM glucose but deprived of nitrogen and sulfate source. attacked among a total of 16 in the subunit, and the altered The suspension was divided into four 500-ml portions and protein is presumably marked for proteolytic degradation shaken at 200 rpm in 2.5-liter flasks for 4 h. The cell extracts (16-18). The Fe3+ probably dissociates from the protein and were cleared by centrifugation at 20,000 x g for 20 min (4°C) is again reduced to Fe2+ by a system, such as the MFO, at and assayed for enzyme activities. Glycerol dehydrogenase the expense of NADH or its equivalent. The regenerated activity was measured as described above for the reactiva- Fe2+ is ready to attack another protein molecule. The results tion experiments. 1,3-Propanediol oxidoreductase activity from the present study suggest that glycerol dehydrogenase was determined in exactly the same way, except that 1,3- is inactivated in an analogous way, although the target amino propanediol replaced glycerol as the substrate. Ethanol acid need not be histidine. dehydrogenase activity was determined by the method of The model would explain the dependence of in vivo McPhedran et al. (23). Glucose 6-phosphate dehydrogenase inactivation of glycerol dehydrogenase on aerobic metabo- lism of a carbon source (20). Reducing equivalents derived thereby, e.g., reduced menaquinone, might react with 02 to form activated species of oxygen, including superoxide and TABLE 1. Restoration of ascorbate-inactivated glycerol hydrogen peroxide. Hydrogen peroxide also appears as a dehydrogenase by high concentrations of NAD' product of superoxide dismutation (6, 9-11, 26). The arising reducing equivalents can, in addition, maintain iron in the Sp act of glycerol dehydroge- Reactant concn (mM) nase (tpmol min-' mg of ferrous state. The requisite hydrogen peroxide and ferrous protein-') at 30°C iron might also be provided by an MFO-mediated system. To model to Ascorbate assess the relevance of the iron-ascorbate the Glycerol NAD Control treated oxidative modification of glycerol dehydrogenase in vivo, experiments are now in progress to examine the nature of 100 0.6 9.1 4.3 changes in the in vitro- and in vivo-inactivated 100 1.2 8.7 5.4 chemical 100 10 11.0 8.8 proteins. 500 0.6 8.8 4.4 The antagonistic effects of Fe2+ and Mn2+ observed with intact cells point to the importance of controlling the internal a Samples of the purified dehydrogenase (1.25 nmol) were incubated with concentrations of these metals in an aerobic environment. shaking for 2 h in tubes (16-mm diameter) containing 1 mnl of 50 mM HEPES (pH 7.3) and 25 ,uM FeCl3 with or without 50 mM ascorbate. Samples from the Enzymes that are activated by Mn2+ in enteric bacteria two mixtures were assayed in the presence of 1 puM Mn2+ and different include glutamine synthetase (35), the NAD-linked malic concentrations of NAD or glycerol. enzyme (25), citrate lyase (33,), L-1,2-propanediol de- 482 NOTES J. BACTERIOL.

hydrogenase (34), and aerobic superoxide dismutase (6). 11. Hassan, H. M., and I. Fridovich. 1977. Regulation of the Most of these enzymes are known also to interact with Fe2+ synthesis of superoxide dismutase in Escherichia coli. Induction and to be susceptible to oxidative inactivation. Certain by methyl viologen. J. Biol. Chem. 252:7667-7672. metalloenzymes, including superoxide dismutases (1, 8, 14) 12. Hueting, S., T. de Lange, and D. W. Tempest. 1978. Properties and dioxygenases (28), are sensitive to H202 inactivation and regulation of synthesis of the glycerol dehydrogenase pre- when they are sent in Klebsiella aerogenes NCTC 418, growing in chemostat associated with Fe>, but are resistant when culture. FEMS Microbiol. Lett. 4:185-189. they are associated with Mn>. 13. Johnson, E. A., S. K. Burke, R. G. Forage, and E. C. C. Lin. Although the transport of Fe2+ (in contradistinction to 1984. Purification and properties of dihydroxyacetone kinase Fe` which is captured as chelates) has not been studied, from Klebsiella pneumoniae. J. Bacteriol. 160:55-60. Mn2+ is known to be accumulated ih Escherichia coli by a 14. Kanematsu, S., and K. Asada. 1979. Ferric and manganic specific, energy-dependent transport system. Approximately superoxide dismutases in Euglena gracilis. Arch. Biochem. 75% of the intracellular Mn2+ is not firmly bound and is Biophys. 195:535-545. readily exchangeable with external Mn2+. Loss of Mn2+ 15. Laemmli, U. K. 1970. Cleavage of structural proteins during the from the cell can be accelerated by Fe2+, and Fe2+ compet- assembly of the head of bacteriophage T4. Nature (London) itively inhibits 227:680-685. Mn2+ uptake (32). These results suggest a 16. Levine, R. L. 1983. Oxidative modification of glutamine synthe- relationship between the intracellular concentrations of free tase. I. Inactivation is due to loss of one histidine residue. J. Mn>+ and Fe>. In particular, the question might be raised Biol. Chem. 258:11823-11827. whether there exist mechanisms that control intracellular 17. Levine, R. L. 1984. Mixed-function oxidation of histidine resi- concentrations of these metals and metal-binding proteins dues. Methods Enzymol. 107:370-376. according to respiratory conditions. In this context it is of 18. Levine, R. L., C. N. Oliver, R. M. Fulks, and E. R. Stadtman. interest to note that the Fe-superoxide dismutase is pro- 1981. Turnover of bacterial glutamine synthetase: oxidative duced constitutively in E. coli, whereas the Mn-superoxide inactivation precedes proteolysis. Proc. Natl. Acad. Sci. USA dismutase is induced in response to 78:2120-2124. oxygen (9-11, 26). E. 19. Lin, E. C. C. 1976. Glycerol dissimilation and its regulation in coli also synthesizes a number of hemeproteins in response bacteria. Annu. Rev. Microbiol. 30:535-578. to oxygen or other electron acceptors. The possibility that 20. Lin, E. C. C., A. P. Levin, and B. Magasanik. 1960. The effect enteric bacteria control the levels of free Mn+ and Fe> to of aerobic metabolism on the inducible glycerol dehydrogenase adapt their metabolism to respiratory conditions deserves of Aerobacter aerogenes. J. Biol. Chem. 235:1824-1829. exploration. 21. Lin, E. C. C., and B. Magasanik. 1960. The activation of glycerol dehydrogenase from Aerobacter aerogenes by We thank Boris Magasanik for drawing our attention to the monovalent cations. J. Biol. Chem. 235:1820-1823. requirements of oxidative metabolism in the inactivation of both 22. McGregor, W. G., J. Phillips, and C. H. Suelter. 1974. Purifica- glycerol dehydrogenase and glutamine synthetase. tion and kinetic characterization of a monovalent cation- Studies in the Harvard laboratory were supported by Public activated glycerol dehydrogenase from Aerobacter aerogenes. Health Service grant 5-RO1-GM11983 from the National Institute of J. Biol. Chem. 249:3132-3139. 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Biochemistry 18:3604-3610. 3. Cleland, W. W., V. W. Thompson, and R. E. Barden. 1969. 26. Nettleton, C. J., C. Bull, T. 0. Baldwin, and J. 0. Fee. 1984. Isocitrate dehydrogenase (TPN-specific) from pig heart. Meth- Isolation of the Escherichia coli superoxide dismutase gene: ods Enzymol. 13:30-33. evidence that intracellular superoxide concentration does not 4. Forage, R. G., and M. A. Foster. 1982. Glycerol fermentation in regulate oxygen-dependent synthesis of the manganese super- Klebsiella pneumoniae: functions of the coenzyme B12- oxide dismutase. Proc. Natl. Acad. Sci. USA 81:4970-4973. dependent glycerol and diol dehydratases. J. Bacteriol. 27. Orsi, B. A., and W. W. Cleland. 1972. Inhibition and kinetic 149:413-419. mechanism of rabbit muscle glyceraldehyde-3-phosphate dehy- 5. Forage, R. G., and E. C. C. Lin. 1982. dha system mediating drogenase. Biochemistry 11:102-109. aerobic and anaerobic dissimilation of glycerol in Kiebsiella 28. Que, L., Jr., J. Widom, and R. L. 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