Purification and characterization of D(--)- -hydroxybutyrate dehydrogenase from Azospirillum brasilense Cd

Abstract

D ( -)-0-hydroxybutyrate dehydrogenase (BOHB-DH) (EC 1.1.1.30) was puri6ed 991-fold from Azospidlum brasilense Cd. Its specific activity was 5650 units (mg protein)-l min-'. The enzyme is a tetramer, with identical subunits and a total molecular mass of 100 kDa. BOHB-DH is not a glycoprotein. It is acidic and contains six disulphide bonds without free S H groups. Under the assay conditions used, BOHB-DH activity was maximal at pH 8.0 and at 36 OC. The enzyme is an NAD+ oxidoreductase, and is inhibited by NADPH and NADH. It has high affinity for P-hydroxybutyrate: the K, value for the P-hydroxybutyrate substrate is 1 mM. Adenosine phosphates, pyruvate, acetyl-coenzyme A, oxaloacetate and 2-oxoglutarate inhibited purified BOHB-DH.

Energy Taxis Is the Dominant Behavior in Azospirillum brasilense

“…PHB oxidation involves a specific NADH-dependent dehydrogenase (35), which competes with the TCA cycle intermediates for the electron transport system (36). The presence of intracellular PHB in the cells (growth above OD 600 ϭ 0.5) prevented chemotaxis to any chemoattractant (Table 3), while the cells remained fully motile and able to respond to repellents.…”

“…During prolonged growth in a rich medium, A. brasilense cells accumulate PHB as an energy and/or carbon storage material (35)(36)(37). PHB oxidation involves a specific NADH-dependent dehydrogenase (35), which competes with the TCA cycle intermediates for the electron transport system (36).…”

“…b Cells were grown to the late exponential growth stage (OD 600 ϭ 0.8) under high aeration in order to achieve high levels of the intracellular PHB (35,36).…”

“…Similarly, inhibitors of the metabolism of a substrate specifically inhibit chemotaxis toward this substrate. The presence of an intracellular metabolizable substrate, PHB, which competes with metabolizable attractants for the electron transport system (35,36), prevents chemotaxis to any extracellular attractant. (ii) There is a direct correlation between the efficiency of a chemical as a growth substrate and as a chemoeffector: the most efficient growth substrates are the stronger attractants for A. brasilense.…”

Identification and Isolation of Genes Involved in Poly(β-Hydroxybutyrate) Biosynthesis in Azospirillum brasilense and Characterization of a phbC Mutant

“…Enzymes involved in the synthesis, accumulation, and degradation of PHAs in A. brasilense have been examined in detail (36,37,38). It was shown that in contrast to other bacterial species, A. brasilense does not produce copolymers of hydroxyalkanoates but rather only homopolymers of PHB (14).…”