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Journal of Bacteriology, July 2002, p. 4018-4024, Vol. 184, No. 14
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.14.4018-4024.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

In Vivo and In Vitro Studies of Bacillus subtilis Ferrochelatase Mutants Suggest Substrate Channeling in the Heme Biosynthesis Pathway

Ulf Olsson,1 Annika Billberg,1,{dagger} Sara Sjövall,1,{ddagger} Salam Al-Karadaghi,2 and Mats Hansson1*

Department of Biochemistry and,1 Department of Molecular Biophysics, Lund University, 22100 Lund, Sweden2

Received 7 January 2002/ Accepted 26 April 2002

Ferrochelatase (EC 4.99.1.1) catalyzes the last reaction in the heme biosynthetic pathway. The enzyme was studied in the bacterium Bacillus subtilis, for which the ferrochelatase three-dimensional structure is known. Two conserved amino acid residues, S54 and Q63, were changed to alanine by site-directed mutagenesis in order to detect any function they might have. The effects of these changes were studied in vivo and in vitro. S54 and Q63 are both located at helix {alpha}3. The functional group of S54 points out from the enzyme, while Q63 is located in the interior of the structure. None of these residues interact with any other amino acid residues in the ferrochelatase and their function is not understood from the three-dimensional structure. The exchange S54A, but not Q63A, reduced the growth rate of B. subtilis and resulted in the accumulation of coproporphyrin III in the growth medium. This was in contrast to the in vitro activity measurements with the purified enzymes. The ferrochelatase with the exchange S54A was as active as wild-type ferrochelatase, whereas the exchange Q63A caused a 16-fold reduction in Vmax. The function of Q63 remains unclear, but it is suggested that S54 is involved in substrate reception or delivery of the enzymatic product.

* Corresponding author. Mailing address: Department of Biochemistry, Lund University, P.O. Box 124, 22100 Lund, Sweden. Phone: 46 46 2220105. Fax: 46 46 2224534. E-mail: mats.hansson{at}biokem.lu.se.

{dagger} Present address: Department of Pure and Applied Biochemistry, Lund University, 22100 Lund, Sweden.

{ddagger} Present address: Department of Plant Biochemistry, Lund University, 22100 Lund, Sweden.

Journal of Bacteriology, July 2002, p. 4018-4024, Vol. 184, No. 14
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.14.4018-4024.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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