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J. Biol. Chem., Vol. 279, Issue 45, 46644-46651, November 5, 2004

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Bacterial Acetone Carboxylase Is a Manganese-dependent Metalloenzyme^*

Jeffrey M. Boyd, Heather Ellsworth, and Scott A. Ensign

From the Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322-0300

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 µM 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 µM Mn²⁺ had a 1.6-fold higher specific activity and 1.9-fold higher manganese content than cells grown with 0.5 µM Mn²⁺, consistently yielding a stoichiometry of 1.9 manganese/₂₂₂ 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 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.

Received for publication, June 25, 2004 , and in revised form, August 23, 2004.

^* This work was supported by National Institutes of Health Grant GM51805. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Medical College of Wisconsin, Milwaukee, WI 53226.

To whom correspondence should be addressed: Dept. of Chemistry and Biochemistry, 0300 Old Main Hill, Utah State University, Logan, UT 84322-0300. Tel.: 435-797-3969; 435-797-3390; E-mail: ensigns{at}cc.usu.edu.

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