ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 209, No. 2, July, pp. 592-597, 1981 Pathway of Galactitol Catabolism in Klebsiella pneumoniae: Oxidation of L-Galactitol- 1 -phosphate by a NAD-Specific Dehydrogenase JOHN P. MARKWELL’ AND RICHARD L. ANDERSON Department of Biochemistry, Michigan State University, East Lansing, Michigan .&824 Received November 20, 1980 The second reaction in the proposed pathway for galactitol catabolism in KlebsieUu penumoniae, oxidation of L-galactitol-l-phosphate to D-tagatose-6-phosphate, is cata- lyzed by an L-galactitol-l-phosphate dehydrogenase activity. This enzyme was purified &fold to remove the other three enzymes of this pathway. Partially purified L-galactitol- l-phosphate dehydrogenase was specific for NAD and the apparent K, values for the substrates were as follows: L-galactitol-l-phosphate, 0.42 mM; NAD+. 0.22 mM; D-tagatose- B-phosphate, 0.10 mM; and NADH. 0.025 mM. The activity was unaffected by monovalent cations, whereas activity and stability exhibited a requirement for divalent cations. The molecular weight of the enzyme was estimated by gel Altration to be 36,600. The equi- librium constant of the reaction forming D-tagatose-6-phosphate from L-galactitol-l- phosphate was determined to be 8.0 X lo-” M. All data are consistent with this activity functioning during galactitol catabolism in tivo. A previous report (1) provided enzy- matic and genetic evidence that KlebsieUa pneumoniae contains several activities in- volved in the catabolism of galactitol. The proposed pathway involves the following compounds as intermediates: galactitol- L-galactitol-1-P --t D-tagatose-6-P - D-ta- gatose-1,6-Pz -+ D-glyeeraldehyde-3-P, and dihydroxyacetone-P. The enzymes of this pathway are a membrane-involved phos- phoenolpyruvate:galactitol phosphotrans- ferase system, L-galactitol-1-P dehydro- genase, D-tagatose-6-P kinase, and D-tag- atose-1,6-Pz aldolase. A similar pathway has been proposed to occur in Escherichia coli (2-4). The phosphotransferase and dehydrogenase activities would seem to be unique to the galactitol catabolic pathway whereas the kinase and aldolase activities have also been demonstrated to partici- i Public Health Service Predoctoral Trainee, Grant GM 01091 from the National Institute of General Medical Sciences. To whom all reprint requests and correspondence should be Pddressed: Department of Biology, University of California, Los Angeles, Calif. 90024. pate in the catabolism of D-g&iCtOSe in some organisms (5, 6). The existence of an L-galactitol-1-P de- hydrogenase in E. coli was suggested 25 years ago (7) and recently confirmed (2). Lengeler has recently reported on some of its characteristics in the cell extract with- out purification (4). In the present report we describe the partial purification of the L-galactitol-1-P dehydrogenase from K. pneumoniae. The other enzymes of the proposed pathway for galactitol catabo- lism were no longer present in the purified fraction. The characteristics of the L-gal- actitol-1-P dehydrogenase were deter- mined to substantiate its proposed role in catabolism of galactitol. MATERIALS AND METHODS The bacterial strain used was Klebsiella pneumo- niae PRL-R3 (formerly designated Ae~obacter aero- genes PRL-R3). The growth of cells and preparation of extracts were as previously described (B), except that the carbon source in the mineral medium was 0.5% (w/v) galactitol unless otherwise indicated. Cul- ture volumes of 1.0 liters were used except for in- 0003-9361/81/030592-06$02.00/0 Copyright 8 1991 by Academic Press. Inc. All rights of reproduction in any form reserved. 592