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Journal of Bacteriology, September 2009, p. 5832-5837, Vol. 191, No. 18
0021-9193/09/$08.00+0 doi:10.1128/JB.00599-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Crystal Structures of Streptococcus suis Mannonate Dehydratase (ManD) and Its Complex with Substrate: Genetic and Biochemical Evidence for a Catalytic Mechanism
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Qiangmin Zhang,1,2
Feng Gao,1
Hao Peng,1
Hao Cheng,1,2
Yiwei Liu,1
Jiaqi Tang,3
John Thompson,4
Guohua Wei,5
Jingren Zhang,6
Yuguo Du,5
Jinghua Yan,1 and
George F. Gao1,2,7*
CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China,1 Graduate University, Chinese Academy of Sciences, Beijing 100049, China,2 Department of Epidemiology, Research Institute for Medicine of Nanjing Command, Nanjing 210002, China,3 Microbial Biochemistry and Genetics Unit, Oral Infection and Immunity Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland 20892,4 Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China,5 Center For Immunology and Microbial Disease, Albany Medical College, Albany, New York,6 Beijing Institutes of Life Science, Chinese Academy of Sciences, Lincui East Road, Beijing 100101, China7
Received 7 May 2009/ Accepted 12 July 2009
Mannonate dehydratase (ManD) is found only in certain bacterial species, where it participates in the dissimilation of glucuronate. ManD catalyzes the dehydration of D-mannonate to yield 2-keto-3-deoxygluconate (2-KDG), the carbon and energy source for growth. Selective inactivation of ManD by drug targeting is of therapeutic interest in the treatment of human Streptococcus suis infections. Here, we report the overexpression, purification, functional characterization, and crystallographic structure of ManD from S. suis. Importantly, by Fourier transform mass spectrometry, we show that 2-KDG is formed when the chemically synthesized substrate (D-mannonate) is incubated with ManD. Inductively coupled plasma-mass spectrometry revealed the presence of Mn2+ in the purified protein, and in the solution state catalytically active ManD exists as a homodimer of two 41-kDa subunits. The crystal structures of S. suis ManD in native form and in complex with its substrate and Mn2+ ion have been solved at a resolution of 2.9 Å. The core structure of S. suis ManD is a TIM barrel similar to that of other members of the xylose isomerase-like superfamily. Structural analyses and comparative amino acid sequence alignments provide evidence for the importance of His311 and Tyr325 in ManD activity. The results of site-directed mutagenesis confirmed the functional role(s) of these residues in the dehydration reaction and a plausible mechanism for the ManD-catalyzed reaction is proposed.
* Corresponding author. Mailing address: Institute of Microbiology, Chinese Academy of Sciences, Datun Road, Chaoyang District, Beijing 100101, People's Republic of China. Phone: 86 10 64807688. Fax: 86 10 64807882. E-mail: gaof{at}im.ac.cn
Published ahead of print on 17 July 2009.
Supplemental material for this article may be found at http://jb.asm.org/.
Journal of Bacteriology, September 2009, p. 5832-5837, Vol. 191, No. 18
0021-9193/09/$08.00+0 doi:10.1128/JB.00599-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
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