In the aconitase superfamily, which includes the archetypical aconitase, homoaconitase, and isopropylmalate isomerase, only aconitase X is not functionally annotated. The corresponding gene (LhpI) was often located within the bacterial gene cluster involved in L-hydroxyproline metabolism. Screening of a library of (hydroxy)proline analogues revealed that this protein catalyzes the dehydration of cis-3-hydroxy-L-proline to Δ1-pyrroline-2-carboxylate. Furthermore, electron paramagnetic resonance and site-directed mutagenic analyses suggests the presence of a mononuclear Fe(III) center, which may be coordinated with one glutamate and two cysteine residues. These properties were significantly different from those of other aconitase members, which catalyze the isomerization of α- to β-hydroxy acids, and have a [4Fe-4S] cluster-binding site composed of three cysteine residues. Bacteria with the LhpI gene could degrade cis-3-hydroxy-L-proline as the sole carbon source, and LhpI transcription was up-regulated not only by cis-3-hydroxy-L-proline, but also by several isomeric 3- and 4-hydroxyprolines.

Hydroxyprolines, such as trans-4-hydroxy-L-proline (T4LHyp), trans-3-hydroxy-L-proline (T3LHyp), and cis-3-hydroxy-L-proline (C3LHyp), are present in some proteins including collagen, plant cell wall, and several peptide antibiotics. In bacteria, genes involved in the degradation of hydroxyproline are often clustered on the genome (L-Hyp gene cluster). We recently reported that an aconitase X (AcnX)-like hypI gene from an L-Hyp gene cluster functions as a monomeric C3LHyp dehydratase (AcnX Type I ). However, the physiological role of C3LHyp dehydratase remained unclear. We here demonstrate that Azospirillum brasilense NBRC 102289, an aerobic nitrogen-fixing bacterium, robustly grows using not only T4LHyp and T3LHyp but also C3LHyp as the sole carbon source. The small and large subunits of the hypI gene (hypI S and hypI L , respectively) from A. brasilense NBRC 102289 are located separately from the L-Hyp gene cluster and encode a C3LHyp dehydratase with a novel heterodimeric structure (AcnX Type IIa ). A strain disrupted in the hypI S gene did not grow on C3LHyp, suggesting its involvement in C3LHyp metabolism. Furthermore, C3LHyp induced transcription of not only the hypI genes but also the hypK gene encoding Δ 1 -pyrroline-2-carboxylate reductase, which is involved in T3LHyp, D-proline, and D-lysine metabolism. On the other hand, the L-Hyp gene cluster of some other bacteria contained not only the AcnX Type IIa gene but also two putative proline racemase-like genes (hypA1 and hypA2). Despite having the same active sites (a pair of Cys/Cys) as hydroxyproline 2-epimerase, which is involved in the metabolism of T4LHyp, the dominant reaction by HypA2 was clearly the dehydration of T3LHyp, a novel type of T3LHyp dehydratase that differed from the known enzyme (Cys/Thr). L-hydroxyproline) degradation in aerobic bacteria, its genetic and molecular information has only recently been elucidated. L-Hydroxyproline metabolic genes are often clustered on bacterial genomes. These loci frequently contain a hypothetical gene(s), whose novel enzyme functions are related to the metabolism of trans-3-hydroxyL-proline and/or cis-3-hydroxyL-proline, a relatively rare L-hydroxyproline in nature. Several L-hydroxyproline metabolic enzymes show no sequential similarities, suggesting their emergence by convergent evolution. Furthermore, transcriptional regulation by trans-4-hydroxy-L-proline, trans-3-hydroxy-L-proline, and/or cis-3-hydroxy-L-proline significantly differs between bacteria. The results of the present study show that several L-hydroxyprolines are available for bacteria as carbon and energy sources and may contribute to the discovery of potential metabolic pathways of another hydroxyproline(s). IMPORTANCE More than 50 years after the discovery of trans-4-hydroxy-L-proline (generally calledKEYWORDS aconitase X, proline racemase superfamily, gene cluster, hydroxyproline, convergent evolution