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

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 ␤-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 ␤-strands. The enzyme binds Zn 2؉ , which is coordinated by Glu155, Asp185, His211, and Glu251. We identified a phosphate-binding site formed by residues from the ␤1/␣1 loop and ␣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 ␤7 and ␤8. 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.It has been known for nearly 70 years that various bacteria, including Escherichia coli, can ferment L-ascorbate (vitamin C) under anaerobic conditions (2,10,30,40). This process has been demonstrated to involve proteins encoded by two divergently transcribed operons, the ulaGR and ulaABCDEF operons (3,40,44). The ulaG gene encodes L-ascorbate-6-phosphate lactonase. The ulaABCDEF operon encodes an L-ascorbate phosphotransferase transport system (UlaABC, formerly designated SgaTBA) responsible for the uptake and phosphorylation of L-ascorbate, and the ulaDEF genes encode three enzymes that catalyze the conversion of the phosphorylated, hydrolytic product of L-ascorbate, 3-keto-L-gulonate-6-phosphate, to D-xylulose-5-phosphate, which is subsequently metabolized by the pentose phosphate pathway. UlaD (formerly SgaH) decarboxylates 3-keto-L-gulonate-6-phosphate, yielding L-xylulose-5-phosphate, which is subsequently epimerized by UlaE (formerly SgaU) at C-3 to L-ribulose-5-phosphate and then epimerized by UlaF (formerly SgaE) at C-4 to D-xylulose-5-phosphate (Fig.

Molecular Underpinnings of Fe(III) Oxide Reduction by Shewanella Oneidensis MR-1

“…MtrC and OmcA are two outer-membrane c -Cyts located on the bacterial surface where they are translocated across the outer-membrane by the bacterial type II secretion system (DiChristina et al, 2002; Myers and Myers, 2003; Donald et al, 2008; Shi et al, 2008; Lower et al, 2009; Reardon et al, 2010). Following expression in S. oneidensis MR-1, OmcA is co-isolated with MtrC when they are solubilized with n -octyl-β- d -glucopyranoside.…”

“…Likewise, MtrC and OmcA are released to the growth medium and are also involved in the formation of chromium [Cr(III)] precipitates that are found in the extracellular matrix following reduction of Cr(VI) by S. oneidensis MR-1 (Shi et al, 2008; Belchik et al, 2011). Global proteomic and Western blot analyses show that the homologs of MtrC and OmcA are the key components of the bound and loosely associated EPS isolated from the biofilm of the metal-reducing bacterium Shewanella sp HRCR-1 (Cao et al, 2011b).…”

Identification and Characterization of MtoA: A Decaheme c-Type Cytochrome of the Neutrophilic Fe(II)-Oxidizing Bacterium Sideroxydans lithotrophicus ES-1

“…In MR-1, MtrA is a decaheme c -Cyt, while MtrB is a trans-outer membrane (OM), porin-like protein. They form a tight protein complex that transfers electrons across the OM to MtrC and OmcA, two OM decaheme c -Cyts that are localized on bacterial outermost surface (Ross et al, 2007; Shi et al, 2008; Hartshorne et al, 2009; Lower et al, 2009; Reardon et al, 2010). MtrABC and OmcA are key components of the MR-1 extracellular electron transfer pathway, which also includes a tetraheme c -Cyt CymA in the inner membrane.…”

The Response of Shewanella oneidensis MR-1 to Cr(III) Toxicity Differs from that to Cr(VI)

“…Reduction and recovery of metals from iron- and manganese-containing minerals by strain MR-1 can involve nanowires (Gorby et al, 2006; El-Naggar et al, 2010) and perhaps also membrane vesicles (Gorby et al, 2008). Genomic analysis indicates that S. oneidensis may produce up to 42 different cytochromes (Meyer et al, 2004), many of which are localized in the outer membrane (Myers and Myers, 2002; Kolker et al, 2005; Bretschger et al, 2007; Shi et al, 2008). Among the latter are the c -type decaheme cytochromes MtrC and OmcA, which occur in a complex (Shi et al, 2006) that is required both for the reduction of many metals (Beliaev and Saffarini, 1998; Beliaev et al, 2001).…”