Biodegradative ornithine decarboxylase of Escherichia coli. Purification, properties, and pyridoxal 5'-phosphate binding site

Biochemistry ◽  
1975 ◽  
Vol 14 (16) ◽  
pp. 3675-3681 ◽  
Author(s):  
Deborah Applebaum ◽  
Donna L. Sabo ◽  
Edmond H. Fischer ◽  
David R. Morris
Keyword(s):  
Escherichia Coli ◽  
Binding Site ◽  
Ornithine Decarboxylase ◽  
Phosphate Binding

scholarly journals 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

2008 ◽  
Vol 190 (24) ◽  
pp. 8137-8144 ◽  
Author(s):  
Rong Shi ◽  
Marco Pineda ◽  
Eunice Ajamian ◽  
Qizhi Cui ◽  
Allan Matte ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Binding Site ◽  
Active Site ◽  
Active Sites ◽  
Triosephosphate Isomerase ◽  
Pentose Phosphate ◽  
Binding Motif ◽  
Phosphate Binding ◽  
Activity Measurements ◽  
Tim Barrel

ABSTRACT 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/α1 loop and α3′ 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.

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