Abstract
Zoogloea ramigera I-16-M was found to contain two stereospecific acetoacetyl CoA reductases; one was NADP+-linked and d(-)-β-hydroxybutyryl CoA specific and the other was NAD+-linked and l(+)-isomer specific. The NADP+-linked enzyme, purified approximately 150-fold, had a pH optimum for the reduction of acetoacetyl CoA at 8.1, but no definite pH optimum for the oxidation of β-hydroxybutyryl CoA. The apparent Michaelis constants for acetoacetyl CoA and NADPH were 8.3 and 21 μM, respectively. The enzyme was markedly inhibited by acetoacetyl CoA at concentrations higher than 10 μM.
The incorporation of [1-14C]acetyl CoA into poly-β-hydroxybutyrate (PHB) by bacterial crude extract (containing β-ketothiolase, acetoacetyl CoA reductases, enoyl CoA hydratases and PHB synthases) or by a system reconstituted from purified preparations of β-ketothiolase, acetoacetyl CoA reductase and PHB synthase, was observed only in the presence of NADPH, but not NADH. Among various enzymes involved in PHB metabolism, only the specific activity of glucose 6-phosphate dehydrogenase was elevated 5-fold within 2 h after the addition of glucose to the cells grown in the basal medium.
These findings suggest that, in Z. ramigera I-16-M, acetoacetyl CoA is directly reduced to d(-)-β-hydroxybutyryl CoA by the NADP+-dependent reductase, and PHB synthesis is at least partially controled by NADPH availability through glucose 6-phosphate dehydrogenase.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Abbreviations
- PHB:
-
poly-β-hydroxybutyrate
References
Bergmeyer, H. U., Gawehn, K., Klotzsch, H., Krebs, H. A., Williamson, D. H.: Purification and properties of crystalline 3-hydroxybutyrate dehydrogenase from Rhodopseudomonas spheroides. Biochem. J. 102, 423–431 (1967)
Cleland, W. W., Thompson, V. W., Barden, R. E.: Isocitrate dehydrogenase (TPN-specific) from pig heart. In: Methods in enzymology, Vol. 13 (J. M. Lowenstein, ed.), pp. 30–33. New York-London: Academic Press 1969
Crabtree, K., McCoy, E., Boyle, W. C., Rohlich, G. A.: Isolation, identification, and metabolic role of the sudanophilic granules of Zoogloea ramigera. Appl. Microbiol. 13, 218–226 (1965)
Flavin, M.: Methylmalonyl coenzyme A. In: Methods in enzymology, Vol. 6 (S. P. Colowick, N. O. Kaplan, eds.), pp. 538–539. New York-London: Academic Press 1963
Fukui, T., Yoshimoto, A., Matsumoto, M., Hosokawa, S., Saito, T., Nishikawa, H., Tomita, K.: Enzymatic synthesis of poly-β-hydroxybutyrate in Zoogloea ramigera. Arch. Microbiol. 110, 149–156 (1976)
Griebel, R., Smith, Z., Merrick, J. M.: Metabolism of poly-β-hydroxybutyrate. I. Purification, composition, and properties of native poly-β-hydroxybutyrate granules from Bacillus megaterium. Biochemistry 7, 3676–3681 (1968)
Joshi, M. D., Jagannathan, V.: Hexokinase. I. Brain. In: Methods in enzymology, Vol. 9 (W. A. Wood, ed.), pp. 371–375. New York-London: Academic Press 1966
Kirkman, H. N.: Glucose 6-phosphate dehydrogenase and human erythrocytes. Nature 184, 1291–1292 (1959)
Layne, E.: Spectrophotometric and turbidimetric methods for measuring proteins. In: Methods in enzymology, Vol. 3 (S. P. Colowick, N. O. Kaplan, eds.), pp. 447–454. New York-London: Academic Press 1957
Lin, L. P., Sadoff, L.: Encystment and polymer production by Azotobacter vinelandii in the presence of β-hydroxybutyrate. J. Bacteriol. 95, 2336–2343 (1968)
Main, R. K., Wilkins, M. J., Cole, L. J.: A modified calcium phosphate for column chromatography of polynucleotides and proteins. J. Am. Chem. Soc. 81, 6490–6495 (1959)
Merrick, J. M., Doudoroff, M.: Enzymatic synthesis of poly-β-hydroxybutyric acid in bacteria. Nature 189, 890–892 (1961)
Moffatt, J. G., Khorana, H. G.: Nucleoside polyphosphates. XII. The total synthesis of coenzyme A. J. Am. Chem. Soc. 83, 663–675 (1961)
Morris, J. G., Redfearn, E. R.: Vitamins and coenzymes. In: Data for biochemical research (R. M. C. Dawson, D. C. Elliot, W. H. Elliot, K. M. Jones, eds.), pp. 192–193. Oxford: Clarendon Press 1969
Moskowitz, G. J., Merrick, J. M.: Metabolism of poly-β-hydroxybutyrate. II. Enzymatic synthesis of d-(-)-β-hydroxybutyryl coenzyme A by an enoyl hydrase from Rhodospirillum rubrum. Biochemistry 8, 2748–2755 (1969)
Nishimura, T., Saito, T., Tomita, K.: Purification and properties of β-ketothiolase from Zoogloea ramigera. Arch. Microbiol. (submitted)
Olsen, I., Merrick, J. M., Goldstein, I. J.: Chemical synthesis of the isomeric dimeric esters of β-hydroxybutyric acid. Biochemistry 4, 453–456 (1965)
Park, J. T., Johnson, M. J.: A submicrodetermination of glucose. J. Biol. Chem. 181, 149–151 (1949)
Ritchie, G. A. F., Senior, P. J., Dawes, E. A.: The purification and characterization of acetoacetyl coenzyme A reductase from Azotobacter beijerinckii. Biochem. J. 121, 309–316 (1971)
Senior, P. J., Dawes, E. A.: The regulation of poly-β-hydroxybutyrate metabolism in Azotobacter beijerinckii. Biochem. J. 134, 225–238 (1973)
Sierra, G., Gibbons, N. E.: Role and oxidation pathway of poly-β-hydroxybutyric acid in Micrococcus halodenitrificans. Can. J. Microbiol. 8, 255–269 (1962)
Simon, E., Shemin, D.: The preparation of S-succinyl coenzyme A. J. Am. Chem. Soc. 75, 2520 (1953)
Stadtman, E. R.: Preparation and assay of acyl coenzyme A and other thiol esters; use of hydroxylamine. In: Methods in enzymology, Vol. 3 (S. P. Colowick, N. O. Kaplan, eds.), pp. 931–941. New York-London: Academic Press 1957
Stanier, R. Y., Doudoroff, M., Kunisawa, R., Contopoulou, R.: The role of organic substrates in bacterial photosynthesis. Proc. Nat. Acad. Sci. U. S. 45, 1246–1260 (1959)
Stern, J. R.: Crystalline crotonase from ox liver. In: Methods in enzymology, Vol. 1 (S. P. Colowick, N. O. Kaplan, eds.), pp. 559–566. New York-London: Academic Press 1955a
Stern, J. R.: Enzymes of acetoacetate formation and breakdown. In: Methods in enzymology, Vol. 1 (S. P. Colowick, N. O. Kaplan, eds.), pp. 573–585. New York-London: Academic Press 1955b
Stokes, J. L., Powers, M. T.: Stimulation of poly-β-hydroxybutyrate oxidation in Sphaerotilus discophorus by manganese and magnesium. Arch. Mikrobiol. 59, 295–301 (1967)
Wieland, T., Rueff, L.: Synthese von S-β-Oxybutyryl- und S-Acetacetyl-Coenzym A. Angew. Chem. 65, 186–187 (1953)
Williamson, D. H., Mellanby, J., Krebs, H. A.: Enzymatic determination of d(-)-β-hydroxybutyric acid and acetoacetic acid in blood. Biochem. J. 82, 90–96 (1962)
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Saito, T., Fukui, T., Ikeda, F. et al. An NADP-linked acetoacetyl CoA reductase from Zoogloea ramigera . Arch. Microbiol. 114, 211–217 (1977). https://doi.org/10.1007/BF00446864
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00446864