Fig 1.
Putative metabolic network of proline and hydroxyproline in archaea.
(A) Metabolic networks of L-proline, D-proline, and T3LHyp. (B) Schematic gene clusters related to the metabolism of proline and/or T3LHyp by archaea. Homologous genes are indicated in the same color and correspond to Fig. 1A. Putative genes in the box were purified and characterized in this study (see Fig. 1C). No clustering was found around the FaProR gene. C-C and C-T indicate a pair of catalytic amino acid residues of ProR superfamily enzymes. Gray putative genes are sequentially similar to other (amino acid) transporters. (C) Purification of recombinant His6 tag proteins. Five micrograms each of the purified protein were applied to a 12% (w/v) gel. (D) Western blot analysis. One microgram each of the purified protein were applied to a 12% (w/v) gel.
Fig 2.
Library of proline derivatives.
TlProR can enable the reversible racemization and epimerization of all proline derivatives (arrows).
Fig 3.
Substrate specificities of TlProR, FaProR, HjProR, CdProR, and AbHypE.
The reaction mixture (1 ml) consisted of 10 mM of substrate and each purified enzyme as follows in Tris-HCl buffer (pH 8.0): TlProR, 1.3 μg; FaProR, 100 μg; HjProR, 570 μg; CdProR, 3.6 μg; AbHypE, 4.9 μg. L-A2C is L-azetidine-2-carboxylate. Samples at the indicated times were analyzed by HPLC (means ± S.D., n = 3). Values on the bars of TlProR indicate specific activity (unit/mg protein), which are similar to those in Tables 1 and 2.
Table 1.
Kinetic parameters for L-proline, T4LHyp, and T3LHyp.
Table 2.
Kinetic parameters for D-proline and C4DHyp.
Fig 4.
Unique inhibition of archaeal ProR-like enzymes by pyrrole-2-carboxylate (PYC; inset).
Reactions were performed for 30 min with the same conditions as those in Fig. 3, except for the presence of several concentration of PYC. L-Proline (for TlProR, FaProR, HjProR, and CdProR) or T4LHyp (for AbHypE) was used as a substrate. Relative specific activity values were expressed as percentages of the values obtained in the absence of PYC (means ± S.D., n = 3). Data for the ProR of T. cruzi (TcProR) are from Berneman et al. [25]. IC50 values were calculated by curve fitting using ImageJ software (http://rsb.info.nih.gov/ij/).
Fig 5.
Representative 1H NMR spectra for epimerase activity toward T4LHyp (A) and T3LHyp (B) by TlProR.
Asterisks are peaks derived from an internal standard. Left panels show the assignments of protons in D2O. The dashed line box indicates the progressive loss of H1 peaks.
Fig 6.
Phylogenetic tree of the ProR superfamily.
The number on each branch indicates the bootstrap value. The circles, squares, and triangles at the end of each branch are enzymes from bacteria, archaea, and eukaryotes, respectively. Proteins with asterisks were used for Fig. 7. Proteins in circles were functionally characterized.
Fig 7.
Partial multiple sequence alignment of deduced amino acid sequences of TlProR.
A, B, and E are enzymes from archaea, bacteria, and eukaryotes, respectively. Consensus segments of the ProR superfamily are shown as a line on the sequence. Catalytic cysteine and/or threonine residues are shaded in red. Gray-shaded letters indicate highly conserved amino acid residues. Seven substrate binding sites are shaded in yellow and green, and the former interact with the carboxyl group or pyrrolidine nitrogen atom. The tryptophan residue, which is important for the discrimination of substrates (Typ241 in TlProR), is shaded in pink. Gray-shaded letters indicate highly conserved amino acid residues. The secondary structures of ProR from T. cruzi (PDB ID 1W61), α-helix (rectangles) and β-sheets (arrows), are shown under the sequence.