Dihydroxyacetone synthase from a methanol-utilizing carboxydobacterium, Acinetobacter sp. strain JC1 DSM 3803

Abstract

Acinetobacter sp. strain JC1 DSM 3803, a carboxydobacterium, grown on methanol was found to show dihydroxyacetone synthase, dihydroxyacetone kinase, and ribulose 1,5-bisphosphate carboxylase, but no hydroxypyruvate reductase and very low hexulose 6-phosphate synthase, activities. The dihydroxyacetone synthase was found to be expressed earlier than the ribulose 1,5-bisphosphate carboxylase. The dihydroxyacetone synthase was purified 19-fold in eight steps to homogeneity, with a yield of 9%. The final specific activity of the purified enzyme was 1.12 mol of NADH oxidized per min per mg of protein. The molecular weight of the native enzyme was determined to be 140,000. Sodium dodecyl sulfate-gel electrophoresis revealed a subunit of molecular weight 73,000. The optimum temperature and pH were 30°C and 7.0, respectively. The enzyme was inactivated very rapidly at 70°C. The enzyme required Mg 2؉ and thiamine pyrophosphate for maximal activity. Xylulose 5-phosphate was found to be the best substrate when formaldehyde was used as a glycoaldehyde acceptor. Erythrose 4-phosphate, glycolaldehyde, and formaldehyde were found to act as excellent substrates when xylulose 5-phosphate was used as a glycoaldehyde donor. The K m s for formaldehyde and xylulose 5-phosphate were 1.86 mM and 33.3 M, respectively. The enzyme produced dihydroxyacetone from formaldehyde and xylulose 5-phosphate. The enzyme was found to be expressed only in cells grown on methanol and shared no immunological properties with the yeast dihydroxyacetone synthase.Carboxydobacteria are a group of facultative chemolithoautotrophic bacteria, except for Streptomyces thermoautotrophicus, which are able to grow aerobically at the expense of carbon monoxide (CO), in addition to several organic materials, as a sole source of carbon and energy (15,24). It has been known for a long time that Pseudomonas gazotropha is the only carboxydobacterium also capable of growing methylotrophically on methanol as the sole carbon and energy sources and is recognized as the first organism adopting three nutrition types, i.e., organotrophy, autotrophy, and methylotrophy (15,25,27,28,39). We, however, observed in a previous study that Acinetobacter sp. strain JC1 DSM 3803, a carboxydobacterium isolated in Korea (6), also grows methylotrophically on both methanol and methylamine as sole carbon and energy sources.It is well known that there are four different ways for assimilation of C 1 compounds in methylotrophic organisms (2). The ribulose monophosphate (RuMP) cycle for the assimilation of formaldehyde, the first metabolite of methanol oxidation, the serine pathway for the fixation of one molecule each of formaldehyde and CO 2 , the end product of methanol oxidation, and the Calvin reductive pentose phosphate cycle for CO 2 fixation were found to work in methylotrophic bacteria, whereas the xylulose monophosphate (XuMP) cycle for the assimilation of formaldehyde was found only in methylotrophic yeasts (2,8,9).In this study, we report the presence of a novel mechanism for the assim...

Properties and functions of the thiamin diphosphate dependent enzyme transketolase

“…Less data are available for DHAS; the enzyme from H. polymorpha, C. boidinii and Acetinobacter sp. displays an even wider range of substrate speci®city, including all of the substrates mentioned for yeast TK, as well as formaldehyde and acetaldehyde as acceptors [70,74,128]. It appears [74] that the Michaelis constants for the donor (Dxylulose 5-phosphate, 1 mM) and acceptor (formaldehyde, 0.43 mM) substrates are similar to those measured for the substrates of TK (Table 1).…”

“…Dihydroxyacetone synthase (DHAS), a special TK present in organisms growing on methanol, has been puri®ed from C. boidinii [74,167] and from the carboxydobacterium Acetinobacter sp. [128], and it is likely to be present in the actinomycete Amycolatopsis methanolica [2].…”

“…Recently, DHAS has been puri®ed from Acetinobacter sp. [128] using a protocol similar to that applied to the puri®cation of this enzyme from C. boidinii [74]. However, the Acetinobacter sp.…”

Growth of Mycobacteria on Carbon Monoxide and Methanol

“…strain JC1 (originally Acinetobacter sp. strain JC1 DSM 3803; reclassified by Song et al [41]), is capable of growing aerobically not only on CO but also on methanol as a sole source of carbon and energy (8,39). This means that the bacterium is able to employ three distinct types of nutrition, chemoheterotrophy, chemolithotrophy, and methylotrophy, depending on substrate availability.…”

Formate-stimulated Oxidation of Methanol byPseudomonas putida9816

“…1) Theˆrst three of these are found in methylotrophic bacteria, and the latter in methylotrophic yeasts. [1][2][3] In 1997, Ro et al 4) reported a methanolutilizing bacteria called Acinetobacter sp. JC1, which relies on the dihydroxyacetone cycle.…”