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Infection and Immunity, February 2006, p. 1222-1232, Vol. 74, No. 2
0019-9567/06/$08.00+0     doi:10.1128/IAI.74.2.1222-1232.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Identification of Amino Acid Residues Involved in Heme Binding and Hemoprotein Utilization in the Porphyromonas gingivalis Heme Receptor HmuR{dagger}

Xinyan Liu,1 Teresa Olczak,2 Hwai-Chen Guo,3 Dabney W. Dixon,4 and Caroline Attardo Genco1*

Department of Medicine and Department of Microbiology, Boston University School of Medicine, Boston, Massachusetts,1 Laboratory of Biochemistry, Institute of Biochemistry and Molecular Biology, Wroclaw University, Wroclaw, Poland,2 Department of Physiology and Biophysics, Boston University School of Medicine, Boston, Massachusetts,3 Department of Chemistry, Georgia State University, Atlanta, Georgia4

Received 9 June 2005/ Returned for modification 8 September 2005/ Accepted 2 November 2005

We have previously identified and characterized a heme/hemoglobin receptor, HmuR, in Porphyromonas gingivalis. To analyze the conserved amino acid residues of HmuR that may be involved in hemin/hemoprotein binding and utilization, we constructed a series of P. gingivalis A7436 hmuR mutants with amino acid replacements and characterized the ability of these mutants to utilize hemin and hemoproteins. Site-directed mutagenesis was employed to introduce mutations H95A, H434A, H95A-H434A, YRAP420-423YAAA, and NPDL442-445NAAA into HmuR in both P. gingivalis and Escherichia coli. Point mutations at H95 and H434 and in the NPDL motif of HmuR resulted in decreased binding to hemin, hemoglobin, and human serum albumin-hemin complex. Notably, mutations of these conserved sites and motifs led to reduced growth of P. gingivalis when human serum was used as the heme source. Analysis using a three-dimensional homology model of HmuR indicated that H95, H434, and the NPDL motif are present on apical or extracellular loops of HmuR, while the YRAP motif is present on the barrel wall. Taken together, these results support a role for H95, H434, and the NPDL motif of the P. gingivalis HmuR protein in heme binding and utilization of serum hemoproteins and the HmuR YRAP motif in serum hemoprotein utilization.

* Corresponding author. Mailing address: Department of Medicine, Section of Infectious Diseases, Boston University School of Medicine, 650 Albany St., Boston, MA 02118. Phone: (617) 414-5305. Fax: (617) 414-5280. E-mail: caroline.genco{at}bmc.org.

{dagger} Supplemental material for this article may be found at http://iai.asm.org/.

Infection and Immunity, February 2006, p. 1222-1232, Vol. 74, No. 2
0019-9567/06/$08.00+0     doi:10.1128/IAI.74.2.1222-1232.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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