Bacterial Acetone Carboxylase Is a Manganese-dependent Metalloenzyme

Abstract

Bacterial acetone carboxylase catalyzes the ATP-dependent carboxylation of acetone to acetoacetate with the concomitant production of AMP and two inorganic phosphates. The importance of manganese in Rhodobacter capsulatus acetone carboxylase has been established through a combination of physiological, biochemical, and spectroscopic studies. Depletion of manganese from the R. capsulatus growth medium resulted in inhibition of acetone-dependent but not malate-dependent cell growth. Under normal growth conditions (0.5 M Mn 2؉ in medium), growth with acetone as the carbon source resulted in a 4-fold increase in intracellular protein-bound manganese over malate-grown cells and the appearance of a Mn 2؉ EPR signal centered at g ‫؍‬ 2 that was absent in malate-grown cells. Acetone carboxylase purified from cells grown with 50 M Mn 2؉ had a 1.6-fold higher specific activity and 1.9-fold higher manganese content than cells grown with 0.5 M Mn 2؉ , consistently yielding a stoichiometry of 1.9 manganese/␣ 2 ␤ 2 ␥ 2 multimer, or 0.95 manganese/␣␤␥ protomer. Manganese in acetone carboxylase was tightly bound and not removed upon dialysis against various metal ion chelators. The addition of acetone to malate-grown cells grown in medium depleted of manganese resulted in the high level synthesis of acetone carboxylase (15-20% soluble protein), which, upon purification, exhibited 7% of the activity and 6% of the manganese content of the enzyme purified from acetone-grown cells. EPR analysis of purified acetone carboxylase indicates the presence of a mononuclear Mn 2؉ center, with possible spin coupling of two mononuclear sites. The addition of Mg⅐ATP or Mg⅐AMP resulted in EPR spectral changes, whereas the addition of acetone, CO 2 , inorganic phosphate, and acetoacetate did not perturb the EPR. These studies demonstrate that manganese is essential for acetone carboxylation and suggest a role for manganese in nucleotide binding and activation.Acetone is a toxic molecule that is produced biologically by the fermentative metabolism of certain anaerobic bacteria and from ketone body breakdown in mammals (1, 2). In addition to producing acetone, mammals and microorganisms also metabolize acetone, producing acetoacetate or acetol (1-hydroxyacetone) from the carboxylation or hydroxylation, respectively, of acetone (3-12). Despite more than 60 years of research on the subject and direct evidence for enzymatic conversion of acetone to acetoacetate and acetol, the physiological significance of acetone metabolism in mammals remains unclear (2, 13). The role of acetone metabolism in bacteria is more clearly defined, in that a variety of aerobic and anaerobic bacteria are able to grow using acetone as their primary source of carbon and energy (14).Despite some early evidence suggesting that acetol is an intermediate in aerobic acetone metabolism by some bacteria (4,6,15), it now appears that carboxylation of acetone to acetoacetate is the primary, if not only, reaction by which both aerobic and anaerobic bacteria initiate acetone catabolism ...

Carboxylases in Natural and Synthetic Microbial Pathways

“…This is exemplified best by acetyl-CoA/propionyl-CoA carboxylase, which is the key enzyme in the autotrophic CO 2 fixation pathways of many archaea, whereas in most bacteria, homologs of this enzyme serve mainly in assimilation and/or biosynthesis. Even more notably, in the case of (21,42,70,106). Thus, it can be expected that even more such enzymes will be discovered.…”

“…On the other hand, some of the assimilatory carboxylases share an evolutionary origin. This seems to be the case for acetophenone and acetone carboxylase, which show high sequence similarity and have similar mechanistic features (21,22,70,109). Recently, genes with homology to those of phenylphosphate carboxylase have been identified as important for the anaerobic metabolism of benzene in the iron-reducing culture BF (1) as well as for naphthalene in the sulfate-reducing enrichment culture N47 (20).…”

Acetone and Butanone Metabolism of the Denitrifying Bacterium "Aromatoleum aromaticum" Demonstrates Novel Biochemical Properties of an ATP-Dependent Aliphatic Ketone Carboxylase

“…The enzymes catalyzing acetone carboxylation in these two species were purified and biochemically characterized. They consist of three subunits and exhibit very similar biochemical properties in coupling the carboxylation reaction to ATP hydrolysis without the need for an organic cofactor (see Table 4) (2,22). The available biochemical and sequence data suggest that acetone carboxylase is the prototype of a specific class of carboxylases, which appear to be present in many different bacterial strains on the basis of their genome sequences (22).…”

“…Acetone carboxylation (Acx) was initially characterized in the aerobic acetone-degrading alphaproteobacterium Xanthobacter autotrophicus and in the anoxygenic phototrophic bacterium Rhodobacter capsulatus (1)(2)(3)(21)(22)(23). The enzymes catalyzing acetone carboxylation in these two species were purified and biochemically characterized.…”

Molecular characterization of mesophilic and thermophilic sulfate reducing microbial communities in expanded granular sludge bed (EGSB) reactors

“…Inhibition of IPA and acetone degraders due to the high sulfide concentrations prevailing in the bioreactor (729-809 mg S 2-l À1 ) is a plausible explanation. Manganese limitation due to formation of insoluble MnS with biogenic sulfide might have contributed to accumulation of acetone in the MR. Bacterial acetone decarboxylase has recently been shown to be a manganese-dependent metalloenzyme (Boyd et al 2004). …”