Article Figures & Data
Figures
- FIG. 1.
Major groups of substrates for the activities of UshAEc and UshAYi enzymes. The structures shown are typical substrates hydrolyzed by the known 5′-nucleotidase and apyrase-like (AMP, ADP, and ATP are shown) (A), UDP-sugar hydrolase (UDP-glucose is shown) (B), or dinucleoside polyphosphate hydrolase (Ap2A, Ap3A, and Ap4A are shown) (C) activities and by the novel CDP-alcohol hydrolase activity (CDP-ethanolamine, CDP-glycerol, and CDP-choline are shown) (D) reported in this work.
- FIG. 2.
Recovery of CDP-ethanolamine hydrolase and 5′-nucleotidase activities from Y. intermedia suspensions. CECT 7230 cells were collected from the surfaces of four COS plates and processed as described in Materials and Methods for the purification of endogenous CDP-alcohol hydrolase, except that (i) COS cultures were grown at different temperatures and (ii) after centrifugation to obtain the step 1 supernatant, the cell precipitates were resuspended in 10 ml of buffer tMg with Triton X-100 and were lysed by sonication. Enzyme activities were assayed in the supernatants (white portions of the bars) and in the lysed precipitates (black portions of the bars). The activities are expressed relative to the numbers of cells in the suspensions as estimated by A 600 measurements.
- FIG. 3.
Endogenous CDP-alcohol hydrolase from Y. intermedia. (A) SDS-PAGE analysis of partially purified preparations. The lanes show analyses of purification steps 3 (III) and 4 (IV) shown in Table S1 in the supplemental material. The gels were stained with silver. The arrows on the right mark the protein bands analyzed by PMF. The band identified as belonging to UshAYi is the ∼63-kDa band. (B) Activation by divalent cations. For this experiment, the enzyme (purification step 3 [see Table S1 in the supplemental material]) was dialyzed against 250 volumes of 20 mM Tris-HCl, pH 7.5, for 20 h, with one buffer change. The initial rates of CDP-ethanolamine hydrolysis were measured in 0.1-ml reaction mixtures using the standard assay, except that different divalent chloride salts were used at the indicated concentrations and bovine serum albumin was omitted. Equal amounts of dialyzed enzyme were used for the assay with each divalent cation.
- FIG. 4.
Expression and purification of the recombinant UshAYi protein. (A) Proteins induced by IPTG in E. coli cells transformed with plasmid pGEX-6P-3-Yi-ushA. The cells transformed with the empty vector pGEX-6P-3 (lanes 1 and 2) or with pGEX-6P-3-Yi-ushA (lanes 3 and 4), either with (+) or without (−) IPTG induction, were lysed and centrifuged. Shown is the SDS-PAGE analysis (Coomassie blue staining) of 12-μl (lanes 1 and 2) or 8-μl (lanes 3 and 4) samples of lysate supernatants. The arrows point to the three proteins induced by IPTG, two of which are specific to cells transformed with the ushA gene of Y. intermedia. The ∼80-kDa band may correspond to the 87.8-kDa GST-UshAYi fusion protein. The ∼60-kDa band may correspond to the UshAYi protein, either including (60.3 kDa) or not including (58.0 kDa) the predicted signal sequence (see Fig. S1 in the supplemental material). (B) Copurification of the activities overexpressed from pGEX-6P-3-Yi-ushA with an ∼60-kDa protein band but not with the GST-UshAYi fusion protein. Shown are (G-75) the chromatographic profiles of purification step 2, including enzyme activities (▪, 5′-nucleotidase on AMP; •, CDP-ethanolamine hydrolase), A 280 (▴), and SDS-PAGE analyses of selected fractions as indicated and (QS) the analysis of the enzymatically active fractions after purification step 3 (see Table S3 in the supplemental material). The arrows mark the gel positions of the ∼80-kDa band (putative GST-UshAYi fusion protein) and the ∼60-kDa band. The PMF of the latter demonstrated unambiguously the presence of the UshAYi protein (not shown).
Tables
- TABLE 1.
Substrate specificities of the endogenous CDP-alcohol hydrolase of Y. intermedia (UshAYi) and overexpressed UshAEc
Substrate Activitya UshAYib UshAEcc AMP 302 169 ADP 146-291d 153-306d ATP 118-353d 101-303d CMP 213 139 CDP 105-210d 139-278d GMP 388 UMP 147 l-Glycerol-3-phosphate <1 <1 d-Glucose-1-phosphate <1 <1 Phosphoethanolamine <1 <1 Phosphocholine <1 <1 2′-AMP <1 <1 CDP-glycerol 237 137 CDP-ethanolamine 100 100 CDP-choline 28 64 CDP-glucose 4 1 UDP-galactose 55 24 UDP-N-acetylglucosamine 40 48 UDP-glucose 38 20 UDP-glucuronate 36 16 UDP-N-acetylgalactosamine 36 18 UDP-galacturonate 10 3 ADP-glucose <1 <1 dTDP-glucose 3 1 GDP-glucose <1 <1 GDP-mannose <1 <1 ADP-ribose 33 5 Ap2A 3 2 Ap3A 12 2 Ap4A <1 <1 Ap5A <1 <1 NAD+ <1 2 NADH <1 1 FAD <1 2 bis-p-Nitrophenylphosphate 4 1 Glycerophosphocholine 2 <1 Glycerophosphoethanolamine <1 <1 4-Nitrophenylphosphocholine <1 <1 4-Nitrophenyl-dTMP <1 <1 2′,3′-cyclic AMP <1 1 3′,5′-cyclic AMP <1 <1 ↵ a Percentage of that measured with CDP-ethanolamine.
↵ b Assayed with enzyme from purification step 3 (see Table S1 in the supplemental material).
↵ c Overexpressed activity in lysate supernatants of E. coli BL21 cells transformed with the cloned ushA gene of E. coli (plasmid pLM-2) (29).
↵ d Assuming that the number of Pi moles formed per mole of substrate split was 1 to 3 (ATP) or 1 to 2 (ADP and CDP).
- TABLE 2.
Hydrolytic activities overexpressed in E. coli cells transformed with ushA Yi
Substrate Activity (U/mg) pGEX-6P-3 pGEX-6P-3-Yi-ushA Overexpresseda AMP 0.08 18.81 18.73 (427) UDP-glucose 0.08 2.15 2.07 (47) CDP-glycerol 0.09 7.04 6.95 (158) CDP-ethanolamine 0.06 4.45 4.39 (100) CDP-choline 0.09 2.1 2.01 (46) CDP-glucose 0.11 0.26 0.15 (3.4) ADP-ribose 0.02 1.0 0.98 (22.3) Ap3A 0.01 0.56 0.55 (12.5) 2′-AMP 0.07 0.09 0.02 (0.5) 2′,3′-cyclic AMP 0.15 0.13 −0.02 (−0.5) 3′,5′-cyclic AMP 0.09 0.09 0.00 (0.0) ↵ a Percentages are in parentheses.
- TABLE 3.
Kinetic parameters and catalytic efficiencies of UshAYi and UshAEc
Substrate From this worka From others kcat (s−1) Km (μM) kcat/Km (M−1 s−1) kcat/Km (M−1 s−1) UshAYi UshAEc UshAYi UshAEc UshAYi UshAEc UshAEc AMP 735 372 9.6 1.8 7.7 × 107 2.1 × 108 1.0 × 107 b 5.4 × 107 c CDP-glycerol 399 324 11.6 2.9 3.4 × 107 1.1 × 108 CDP-ethanolamine 330 261 8.1 1.7 4.1 × 107 1.5 × 108 CDP-choline 172 231 10.3 2.4 1.7 × 107 9.6 × 107 UDP-glucose 97 71 9.6 10.4 1.0 × 107 6.8 × 106 1.0 × 107 d ADP-ribose 72 5.8 1.2 × 107 Ap3A 1.0 × 107 e ↵ a UshAYi was the recombinant enzyme of Y. intermedia expressed in E. coli and purified (see Table S3 in the supplemental material). UshAEc was the overexpressed enzyme of E. coli BL-21 cells transformed with plasmid pLM-2; the enzyme was purified by the same procedure as for UshAYi, and a nearly homogeneous preparation was obtained (not shown). The standard enzyme assay was used, with a concentrated Pi reagent to measure initial rates at different substrate concentrations. The data were adjusted to the Michaelis-Menten equation (12).
↵ b Calculated as follows, with Co2+ as the activating cation: k cat = 750 s−1 and K m = 74 μM (39).
↵ c Calculated as follows, with Co2+-Ca2+ as the activating cation: k cat = 2,440 s−1 and K m = 45 μM (38).
↵ d Calculated as follows, with Mn2+ as the activating cation: k cat = 504 s−1 and K m = 45 μM (38).
↵ e Calculated as follows, with Mn2+ as the activating cation: k cat = 360 s−1 and K m = 35 μM (38).
Supplemental material
Files in this Data Supplement:
- Supplemental file 1 -
Table S1, purification of endogenous CDP-alcohol hydrolase; Table S2, products of reactions catalyzed by endogenous CDP-alcohol
hydrolase; Table S3, purification of Yi-UshA; Fig. S1, alignment of UshA from Y. intermedia and E. coli; Fig. S2, theoretical structures of Yi-UshA; Fig. S3, conservation in Yi-UshA of the spatial distribution of amino acids.
PDf file, 1MB.
- Supplemental file 1 -
Table S1, purification of endogenous CDP-alcohol hydrolase; Table S2, products of reactions catalyzed by endogenous CDP-alcohol
hydrolase; Table S3, purification of Yi-UshA; Fig. S1, alignment of UshA from Y. intermedia and E. coli; Fig. S2, theoretical structures of Yi-UshA; Fig. S3, conservation in Yi-UshA of the spatial distribution of amino acids.
