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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).

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