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. 2003 Oct 3;332(5):1083-94.
doi: 10.1016/j.jmb.2003.08.009.

The 2.2 A resolution structure of RpiB/AlsB from Escherichia coli illustrates a new approach to the ribose-5-phosphate isomerase reaction

Rong-Guang Zhang  1 , C Evalena Andersson, Tatiana Skarina, Elena Evdokimova, Aled M Edwards, Andrzej Joachimiak, Alexei Savchenko, Sherry L Mowbray
Affiliations

The 2.2 A resolution structure of RpiB/AlsB from Escherichia coli illustrates a new approach to the ribose-5-phosphate isomerase reaction

Rong-Guang Zhang et al. J Mol Biol. .

Abstract

Ribose-5-phosphate isomerases (EC 5.3.1.6) interconvert ribose 5-phosphate and ribulose 5-phosphate. This reaction permits the synthesis of ribose from other sugars, as well as the recycling of sugars from nucleotide breakdown. Two unrelated types of enzyme can catalyze the reaction. The most common, RpiA, is present in almost all organisms (including Escherichia coli), and is highly conserved. The second type, RpiB, is present in some bacterial and eukaryotic species and is well conserved. In E.coli, RpiB is sometimes referred to as AlsB, because it can take part in the metabolism of the rare sugar, allose, as well as the much more common ribose sugars. We report here the structure of RpiB/AlsB from E.coli, solved by multi-wavelength anomalous diffraction (MAD) phasing, and refined to 2.2A resolution. RpiB is the first structure to be solved from pfam02502 (the RpiB/LacAB family). It exhibits a Rossmann-type alphabetaalpha-sandwich fold that is common to many nucleotide-binding proteins, as well as other proteins with different functions. This structure is quite distinct from that of the previously solved RpiA; although both are, to some extent, based on the Rossmann fold, their tertiary and quaternary structures are very different. The four molecules in the RpiB asymmetric unit represent a dimer of dimers. Active-site residues were identified at the interface between the subunits, such that each active site has contributions from both subunits. Kinetic studies indicate that RpiB is nearly as efficient as RpiA, despite its completely different catalytic machinery. The sequence and structural results further suggest that the two homologous components of LacAB (galactose-6-phosphate isomerase) will compose a bi-functional enzyme; the second activity is unknown.

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Figures

Figure 1
Figure 1
Ribose-5-phosphate isomerase reaction. The expected catalytic acid and base are placed with respect to the substrates, products and intermediates; carbon atoms of the sugars are numbered. In the first step with ribose 5-phosphate as substrate, acid–base catalysis provided by either solvent or the enzyme opens the five-membered furanose ring to give the open-chain aldose. A basic group of the enzyme must then extract a proton from C2 of the aldose, generating a cis-1,2-enediol(ate) intermediate, and release the same proton at C1. A second protein group located on the opposite site of the active site transfers a proton from O2 to O1, to produce the ketose sugar.
Figure 2
Figure 2
Enzymatic characterization. Data from a typical series of kinetic experiments using the second construct are shown, together with the curve calculated from the Michaelis–Menten equation using the overall average kcat of 69 s−1 and Km of 1.23 mM (enzyme concentration 30 nM). The inset presents the same data in a Hanes–Woolf plot; the line calculated with the average kinetic constants is shown here in gray.
Figure 3
Figure 3
Electron density. Representative section of electron density in the original MAD map calculated at 2.4 Å resolution (contoured at 1σ), shown together with the structure of the final refined model.
Figure 4
Figure 4
Overall structure of RpiB. (a) The tetramer of the asymmetric unit is shown; the subunits of the A/D dimer are violet and blue, respectively, while those of the B/C dimer are orange and yellow; in all cases a darker shade indicates the central β-sheet of the subunit. (b) The AD dimer is shown, with each chain color-coded, beginning with blue at the N terminus and going through the rainbow to red at the C terminus. (c) Topology diagram of a subunit, colored in the same way as in (b). The first and last residues of secondary structural elements are indicated, along with the naming convention. Residues lining the active site are indicated as magenta stars when they originate from one subunit, and pink stars if they originate from the second of the dimer. The N-terminal His-tag is omitted in (b) and in (c).
Figure 5
Figure 5
Sequence conservation in the RpiB/LacAB family. Eleven representative sequences are aligned with that of E. coli K12 RpiB (gi:16131916), and shaded according to the degree of conservation: Yersinia pestis (gi:16123503), Bacillus subtilis (gi:16080745), Aquifex aeolicus (gi:15606395), Thermotoga maritima (gi:15643838), Rickettsia conorii (gi:15892325), Helicobacter pylori J99 (gi:15611588), Giardia lamblia ATCC 50803 (gi:29248748), Chlorobium tepidum TLS (gi:21673876), Mycoplasma pulmonis (gi:15829083), Clostridium acetobutylicum (gi:15896134) and Mycobacterium tuberculosis H37Rv (gi:15609602). Residues are annotated if they lie within the RpiB active-site pocket (boldface a for one subunit, plaintext a for the second), or in the dimer (d) or tetramer (t) interfaces. In several cases where a residue is involved in both dimer and tetramer contacts, only the dimer interaction is indicated. Four additional sequences represent proteins with documented LacA and LacB function: Lactococcus lactis lacB (gi:149407); Staphylococcus aureus LacB (gi:21283849); L. lactis LacA (gi:149406); S. aureus LacA (gi:21283850). Residues in the active-site pocket that are conserved within these subfamilies are highlighted as follows: those that are similar to RpiB in sequence are shown in yellow, and those that suggest a different function are shown in gray.
Figure 6
Figure 6
Conservation of the molecular surfaces. (a) The surface of a subunit showing sequence conservation (based on the RpiB alignments shown in Figure 5), color-coded going from yellow (least conserved) to red (most conserved). The residues of the two halves of the putative active site are labeled. (b) The dimer surface color-coded in the same way.
Figure 7
Figure 7
Active sites of ribose-5-phosphate isomerases. (a) Close-up view of the active site of RpiB. One subunit is colored according to sequence as in Figure 4(c), the other is colored gray; residues are indicated with if they are contributed by the second subunit in the dimer. (b) The active site of RpiA bound to the inhibitor, arabinose 5-phosphate (PDB entry 1O8B; sugar shown with carbon atoms black, oxygen atoms gray, and phosphate ion salmon).
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