For the ornithine fermentation pathway, described more than 70 years ago, genetic and biochemical information are still incomplete. We present here the experimental identification of the last four missing genes of this metabolic pathway. They encode l-ornithine racemase, (2R,4S)-2,4-diaminopentanoate dehydrogenase, and the two subunits of 2-amino-4-ketopentanoate thiolase. While described only for the Clostridiaceae to date, this pathway is shown to be more widespread.

Since genes coding for enzymes involved in the same metabolic pathway are often colocalized on the bacterial chromosome, the genomic context of oraS and oraE (the only known genes of the pathway) was explored in the following: (i) the ~1,100 sequenced bacterial genomes, using the Integrated Microbial Genomes interface (http://img.jgi.doe.gov); (ii) the assembled contigs from a metagenomics resource from the anaerobic digester of a wastewater treatment plant (12); and (iii) the genome of C. sticklandii, which was recently sequenced in our laboratory. Analyses of the latter two resources were performed using the microbial genome annotation system MaGe (19). The genomic contexts of bacterial genomes and a metagenomic sequence possessing the oraS and oraE genes are shown in Fig. ​Fig.2.2. At least five genes, including oraS and oraE, are found in synteny (from or-1 to oraE). Moreover, the synteny conservation is extended to two additional genes (or-4 and or-5) in eight of the genomes. This conserved genomic neighborhood suggests that the genes flanking oraS and oraE could be involved in the oxidative degradation of l-ornithine. DAPDH (Fig. ​(Fig.1,1, step 3) from C. sticklandii has been described as a homodimer with a subunit molecular mass of about 36 kDa (13). Interestingly, one of the genes of the cluster, or-1 (Fig. ​(Fig.2),2), encodes a protein with a computed molecular mass of 38 kDa. This protein contains a predicted N-terminal NAD(P)⁺-binding Rossmann fold, consistent with dehydrogenase activity. These data therefore support or-1 as a promising candidate. Concerning AKPT (Fig. ​(Fig.1,1, step 4), the only available biochemical data concern its stimulation by PLP (9). The protein encoded by the gene or-3 (Fig. ​(Fig.2)2) presents (weak) similarities with PLP-dependent enzymes, such as threonine, tryptophan, and cysteine synthases. Moreover, the InterProScan software program classifies this protein as a “tryptophan synthase β-subunit-like PLP-dependent enzyme.” Therefore, the or-3 gene seemed to be an interesting candidate for encoding the AKPT activity. Nevertheless, the conserved presence of a small gene (or-2) just upstream of the AKPT gene candidate suggests by analogy with tryptophan synthase that AKPT may be composed of two different subunits encoded by genes or-2 and or-3. Finally, OR (Fig. ​(Fig.1,1, step 1) has been described by Chen et al. as a PLP-dependent enzyme with a subunit molecular mass of 46.8 kDa (2). The or-5 gene is present in the conserved gene cluster in eight organisms, including C. sticklandii (Fig. ​(Fig.2),2), and is predicted to encode a putative amino acid/alanine racemase. Its homology with known alanine racemases is weak but reaches 26% in the N-terminal region, which includes the PLP fixation site. However, the alanine racemase consensus PLP attachment site (prosite) is not present, and the C-terminal part of the or-5 protein shares no similarity with known racemases. This suggests that or-5 may encode a new type of racemase. In spite of the slight size difference between the protein encoded by or-5 and the one characterized by Chen et al. (39.4 versus 46.8 kDa), it was nevertheless considered the most promising candidate for OR activity.

The first attempts to characterize AKPT activity were conducted with the product of or-3. Since no enzymatic activity could be detected, it was considered that AKPT might be composed of subunits encoded by distinct genes and acting as a complex. Because or-2 is always found next to the AKPT candidate gene (or-3), it was expected that these two genes could encode the α and β subunits of AKPT, respectively. The two genes were then cloned as a polycistronic sequence in pEXP5-NT/TOPO for recombinant production. Under these conditions, the hexahistidine tag was inserted only into the putative α subunit of the complex. SDS-PAGE analysis after immobilized metal affinity chromatography revealed the presence of two purified polypeptide chains with apparent molecular masses around 14 and 50 kDa (not shown), consistent with the calculated masses of 13,930 and 51,450 Da for the putative α and β subunits, respectively. The copurification of the polypeptide chain deprived of the hexahistidine sequence using an Ni²⁺ affinity column strengthens the hypothesis that AKPT is composed of distinct subunits. But according to SDS-PAGE analysis, the absence of a His-tagged sequence on the putative β subunit led to its substoichiometric proportion. To purify the α and β subunits in equimolar proportions, gel filtration experiments were therefore conducted on the mixture containing the two purified proteins. A total of 300 μg of putative AKPT in 50 mM Tris-HCl (pH 8.0)-150 mM NaCl was loaded onto the column. A peak with an apparent mass of 136 kDa, consistent with a functional heterotetrameric α₂β₂ structure, was isolated. SDS-PAGE analysis revealed that the protein fractions recovered contained both α and β subunits in apparent stoichiometric proportions and purified to apparent homogeneity (not shown). AKPT activity was thus assayed in the forward direction, following the production of acetyl-CoA (Fig. ​(Fig.1,1, step 4). The formation of the thioester bond of acetyl-CoA could be continuously monitored (₂₃₂ = 4,500 M⁻¹·cm⁻¹) (15). The reaction was carried out in 50 mM Tris-HCl (pH 8.0) in the presence of 40 μM PLP, 80 μM CoA-SH, and 1 mM AKP, confirming the functionality of the purified thiolase. Experiments indicated that, in agreement with the data of Jeng et al. (9), AKPT activity is strictly PLP dependent (not shown). The kinetic constants of AKPT for AKP and CoA-SH were evaluated by varying the concentration of one substrate while keeping the other substrate at the saturating concentration. The kinetic parameters were obtained from duplicate experiments by nonlinear analysis of initial rates. Results are presented in Table ​Table1.1. The identity of the compound detected at 232 nm was further analyzed using citrate synthase. Briefly, AKP was allowed to react with CoA-SH in the presence of AKPT to accumulate acetyl-CoA. Twenty-five micrograms of AKPT was incubated in 100 μl 50 mM Tris-HCl (pH 8.0) in the presence of 40 μM PLP, 80 μM CoA-SH, and 1 mM AKP for 1 h. The reaction was stopped by the addition of 1% (vol/vol) trifluoroacetic acid and neutralized with 5 M K₂CO₃, and the mixture was further incubated with 450 μM dithionitrobenzoate to deplete free CoA-SH. The depletion of the free CoA-SH pool was continuously monitored at 412 nm through the formation of the thionitrobenzoate ion until a plateau was reached. The addition of 0.4 U of citrate synthase and 1 mM oxaloacetate led to a new strong increase in absorption, confirming the presence of acetyl-CoA synthesized by AKPT. Alanine has been described to be the other reaction product, but its conformation has never been established, while it is expected to be the d isomer (9). Indeed, d-ornithine is the substrate for ornithine mutase (14), and there is no evidence for further steric rearrangements of the α-amino group during dehydrogenase and thiolase reactions. The identification of the isomer conformation was experimentally established, taking advantage of the reversibility of the AKPT reaction (Fig. ​(Fig.1,1, step 4). A 2.5-μg amount of AKPT was incubated in 100 μl 50 mM Tris-HCl (pH 8.1), containing 40 μM PLP and 450 μM dithionitrobenzoate, in the presence of 2 mM acetyl-CoA and 2 mM d-alanine. The reaction was initiated by the addition of d-alanine and monitored continuously at 412 nm. The formation of CoA-SH was observed only in the presence of the d isomer as the substrate and not in that of the l isomer (not shown). The substrate specificity of the enzyme was investigated in a similar way. Several amino acids and esters of CoA were tested for thiolase activity. The enzyme was incubated as follows: (i) in the presence of acetyl-CoA and of each proteogenic amino acid (with the exception of cysteine) in its d conformation; (ii) in the presence of d-alanine and of a CoA ester (propionyl-CoA, crotonyl-CoA, succinyl-CoA, butyryl-CoA, or malonyl-CoA). Under these conditions, the formation of CoA-SH was never observed, suggesting a strict substrate specificity. AKPT has no significant homology with any known enzyme family. Homologs of AKPT (>57% identity over the whole length of the protein) were found only in genomes containing all the genes of the pathway, which are expected to ferment this amino acid. In the other organisms, the maximum percentage identity falls to ~26%, with other PLP-dependent enzymes, such as tryptophan synthase.

OR catalyzes the conversion of l- to d-ornithine (Fig. ​(Fig.1,1, step 1). The His-tagged recombinant putative OR has a calculated molecular mass consistent with its apparent mass in SDS-PAGE (41,763.9 Da and ~43 kDa, respectively). The protein was purified as a homodimer by gel filtration (not shown). The UV/visible spectrum of the purified protein revealed an absorption maximum at 420 nm, suggesting the presence of tightly bound PLP (not shown). These data can be linked to the work of Chen et al., who described the purification of dimeric and PLP-dependent OR with an apparent mass of 46.8 kDa on an SDS-PAGE gel for C. sticklandii (2). Here, the OR activity was first demonstrated by NADH formation in a coupled enzymatic assay with excess OA and DAPDH. The pyridine nucleotide reduction was triggered by the addition of l-ornithine, proving the functionality of the enzyme. Preliminary experiments with saturating concentrations of l-ornithine were conducted to set up an assay with adequate amounts of enzymes. Particular care was given to the handling of the racemase due to its instability upon dilution (~40% activity lost within 2 h). Under these conditions, the rate of formation of NADH was strictly proportional to the amount of OR (with amounts of enzyme ranging from 0.24 to 1.43 ng). The coupled assay was conducted using 0.24 ng OR, 20 μg OA, 5.5 μg DAPDH, 2 mM NAD⁺, 3 μM adenosylcobalamine, and 40 μM PLP in 100 μl 50 mM Tris-HCl, pH 8.5. The kinetic parameters of OR were determined from triplicate experiments by nonlinear analysis of initial rates and are shown in Table ​Table1.1. Data are close to those previously reported (2): K_m = 770 μM; k_cat = 980 s⁻¹. To further characterize the enzyme, its substrate specificity was assessed. Five mM of l and d enantiomers of all proteogenic amino acids plus ornithine were incubated in 100 μl 10 mM Tris-HCl (pH 8.5) in the presence of 10 μM PLP and 8 μg OR for 3 h. l and d enantiomers of amino acids were separated on a Sumichiral OA-6100 column (150 by 4.6 mm, 5 μm; Sumika) using an isocratic gradient (1 mM CuSO₄ plus 2% acetonitrile) with a flow rate of 1 ml/min. Compounds were detected by a photodiode array (Thermo-Fisher) between 200 and 600 nm. Results indicated that OR racemizes preferentially basic amino acids; for lysine, arginine, and ornithine, the reaction reached equilibrium with around 50% conversion for each enantiomer. Three other amino acids, serine, asparagine, and alanine, were also converted, but at a lower rate, with a maximum of only 10% serine conversion within the time course of the experiment (data not shown). The conversion of d- to l-alanine was further confirmed by a spectrophotometric assay coupling the formation of l-alanine to NAD⁺ reduction in the presence of l-alanine dehydrogenase. These results are thus in disagreement with the work of Chen et al. (2), where a strict specificity for ornithine was reported. Finally, these discrepancies (the slight difference in molecular mass but especially the marked difference in substrate specificity) suggest that the racemase presented here could be different from the one previously reported.

The nucleotide sequences reported in this article have been submitted to the GenBank/EBI data bank with the accession numbers CU695246, CU695247, CU695248, and CU695250.