Bacterial Acetone Carboxylase Is a Manganese-dependent Metalloenzyme

Jeffrey M Boyd,Heather Ellsworth,Scott A Ensign

doi:10.1074/jbc.m407177200

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 microm Mn2+ 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 Mn2+ EPR signal centered at g = 2 that was absent in malate-grown cells. Acetone carboxylase purified from cells grown with 50 microm Mn2+ had a 1.6-fold higher specific activity and 1.9-fold higher manganese content than cells grown with 0.5 microm Mn2+, consistently yielding a stoichiometry of 1.9 manganese/alpha2beta2gamma2 multimer, or 0.95 manganese/alphabetagamma 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 Mn2+ center, with possible spin coupling of two mononuclear sites. The addition of Mg.ATP or Mg.AMP resulted in EPR spectral changes, whereas the addition of acetone, CO2, 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.

Highlights

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]
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
Despite some early evidence suggesting that acetol is an intermediate in aerobic acetone metabolism by some bacteria [4, 6, 15], it appears that carboxylation of acetone to acetoacetate is the primary, if, reaction by which both aerobic and anaerobic bacteria initiate acetone catabolism [14]

Summary

EXPERIMENTAL PROCEDURES

Growth of Bacteria, and Preparation of Cell Extracts— R. capsulatus strain B10 (ATCC 33303) [26] was grown photoheterotrophically at 30 °C using the media and growth conditions described previously [24], with the following modifications. For purification of acetone carboxylase from manganese-depleted cultures, all buffers were treated with Chelex, and glassware was washed with HNO3 as described above. Acetone carboxylase activity in purified protein samples was determined using gas chromatography as described previously [24, 28]. For EPR and metal analysis of cell lysates, samples were incubated with Chelex 100 for 10 min and passed over a 1.5 ϫ 5-cm column of Sephadex G-25 (PD-10; Amersham Biosciences), which had 1 cm of Chelex overlaid on the resin. For EPR analysis of purified proteins, samples were treated with Chelex 100 followed by centrifugation (1000 ϫ g) and decanting to remove the Chelex resin. Nucleotides, acetone, and other molecules were added to appropriate samples, followed by evacuation, flushing, and transfer to degassed EPR tubes as described above. All samples were treated with Chelex 100 as described above prior to performing metal analysis

RESULTS

Acetone carboxylase sourcea

DISCUSSION