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Journal of Bacteriology, December 2008, p. 8137-8144, Vol. 190, No. 24
0021-9193/08/$08.00+0     doi:10.1128/JB.01049-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Structure of L-Xylulose-5-Phosphate 3-Epimerase (UlaE) from the Anaerobic L-Ascorbate Utilization Pathway of Escherichia coli: Identification of a Novel Phosphate Binding Motif within a TIM Barrel Fold{triangledown}

Rong Shi,1 Marco Pineda,1 Eunice Ajamian,1 Qizhi Cui,2 Allan Matte,2 and Miroslaw Cygler1,2*

Department of Biochemistry, McGill University, 3655 Promenade Sir William Osler, Montréal, Québec H3G 1Y6, Canada,1 Biotechnology Research Institute, NRC, 6100 Royalmount Avenue, Montréal, Québec H4P 2R2, Canada2

Received 28 July 2008/ Accepted 21 September 2008

Three catabolic enzymes, UlaD, UlaE, and UlaF, are involved in a pathway leading to fermentation of L-ascorbate under anaerobic conditions. UlaD catalyzes a β-keto acid decarboxylation reaction to produce L-xylulose-5-phosphate, which undergoes successive epimerization reactions with UlaE (L-xylulose-5-phosphate 3-epimerase) and UlaF (L-ribulose-5-phosphate 4-epimerase), yielding D-xylulose-5-phosphate, an intermediate in the pentose phosphate pathway. We describe here crystallographic studies of UlaE from Escherichia coli O157:H7 that complete the structural characterization of this pathway. UlaE has a triosephosphate isomerase (TIM) barrel fold and forms dimers. The active site is located at the C-terminal ends of the parallel β-strands. The enzyme binds Zn2+, which is coordinated by Glu155, Asp185, His211, and Glu251. We identified a phosphate-binding site formed by residues from the β1/{alpha}1 loop and {alpha}3' helix in the N-terminal region. This site differs from the well-characterized phosphate-binding motif found in several TIM barrel superfamilies that is located at strands β7 and β8. The intrinsic flexibility of the active site region is reflected by two different conformations of loops forming part of the substrate-binding site. Based on computational docking of the L-xylulose 5-phosphate substrate to UlaE and structural similarities of the active site of this enzyme to the active sites of other epimerases, a metal-dependent epimerization mechanism for UlaE is proposed, and Glu155 and Glu251 are implicated as catalytic residues. Mutation and activity measurements for structurally equivalent residues in related epimerases supported this mechanistic proposal.

* Corresponding author. Mailing address: Biotechnology Research Institute, NRC, 6100 Royalmount Avenue, Montréal, Québec H4P 2R2, Canada. Phone: (514) 496-6321. Fax: (514) 496-5143. E-mail: mirek{at}bri.nrc.ca

{triangledown} Published ahead of print on 10 October 2008.

Journal of Bacteriology, December 2008, p. 8137-8144, Vol. 190, No. 24
0021-9193/08/$08.00+0     doi:10.1128/JB.01049-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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