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Identification and characterization of trans-3-hydroxy-l-proline dehydratase and Δ(1)-pyrroline-2-carboxylate reductase involved in trans-3-hydroxy-l-proline metabolism of bacteria.

Watanabe S, Tanimoto Y, Yamauchi S, Tozawa Y, Sawayama S, Watanabe Y - FEBS Open Bio (2014)

Bottom Line: In A. brasilense cells, T3LHyp dehydratase and NAD(P)H-dependent Δ(1)-pyrroline-2-carboxylate (Pyr2C) reductase activities were induced by T3LHyp (and d-proline and d-lysine) but not T4LHyp, and no effect of T3LHyp was observed on the expression of T4LHyp metabolizing enzymes: a hypothetical pathway of T3LHyp → Pyr2C → l-proline was proposed.On the other hand, Pyr2C reductase encoded to LhpI gene was a novel member of ornithine cyclodeaminase/μ-crystallin superfamily, differing from known bacterial protein.Furthermore, disruption of LhpI gene from A. brasilense led to loss of growth on T3LHyp, d-proline and d-lysine, indicating that this gene has dual metabolic functions as a reductase for Pyr2C and Δ(1)-piperidine-2-carboxylate in these pathways, and that the T3LHyp pathway is not linked to T4LHyp and l-proline metabolism.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama, Ehime 790-8566, Japan.

ABSTRACT
trans-4-Hydroxy-l-proline (T4LHyp) and trans-3-hydroxy-l-proline (T3LHyp) occur mainly in collagen. A few bacteria can convert T4LHyp to α-ketoglutarate, and we previously revealed a hypothetical pathway consisting of four enzymes at the molecular level (J Biol Chem (2007) 282, 6685-6695; J Biol Chem (2012) 287, 32674-32688). Here, we first found that Azospirillum brasilense has the ability to grow not only on T4LHyp but also T3LHyp as a sole carbon source. In A. brasilense cells, T3LHyp dehydratase and NAD(P)H-dependent Δ(1)-pyrroline-2-carboxylate (Pyr2C) reductase activities were induced by T3LHyp (and d-proline and d-lysine) but not T4LHyp, and no effect of T3LHyp was observed on the expression of T4LHyp metabolizing enzymes: a hypothetical pathway of T3LHyp → Pyr2C → l-proline was proposed. Bacterial T3LHyp dehydratase, encoded to LhpH gene, was homologous with the mammalian enzyme. On the other hand, Pyr2C reductase encoded to LhpI gene was a novel member of ornithine cyclodeaminase/μ-crystallin superfamily, differing from known bacterial protein. Furthermore, the LhpI enzymes of A. brasilense and another bacterium showed several different properties, including substrate and coenzyme specificities. T3LHyp was converted to proline by the purified LhpH and LhpI proteins. Furthermore, disruption of LhpI gene from A. brasilense led to loss of growth on T3LHyp, d-proline and d-lysine, indicating that this gene has dual metabolic functions as a reductase for Pyr2C and Δ(1)-piperidine-2-carboxylate in these pathways, and that the T3LHyp pathway is not linked to T4LHyp and l-proline metabolism.

No MeSH data available.


(A) Enzyme activities of cell-free extracts prepared from A. brasilense cells grown on several carbon sources. Values are the means ± SD, n = 2. Light- and dark-gray bars in Pyr2C reductase indicate NADPH- and NADH-dependent activities, respectively. (B) The transcriptional effect of carbon source on AbLhpI gene. Total RNAs (4 μg per lane) were isolated from the A. brasiliense cells grown on the indicated carbon sources.
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f0010: (A) Enzyme activities of cell-free extracts prepared from A. brasilense cells grown on several carbon sources. Values are the means ± SD, n = 2. Light- and dark-gray bars in Pyr2C reductase indicate NADPH- and NADH-dependent activities, respectively. (B) The transcriptional effect of carbon source on AbLhpI gene. Total RNAs (4 μg per lane) were isolated from the A. brasiliense cells grown on the indicated carbon sources.

Mentions: First, we found that among three bacteria capable of metabolizing T4LHyp, only A. brasilense can grow on T3LHyp as a sole carbon source, not P. putida and P. aeruginosa; to our knowledge, this is the first report of T3LHyp metabolism by an organism(s) (Figs. 1E and S1). Next, we estimated whether the T4LHyp pathway is related to T3LHyp metabolism because of its structural similarity. However, all four enzymes involved in T4LHyp metabolism were induced only by T4LHyp (and C4DHyp), not by T3LHyp (Fig. 2A). On the other hand, significant activities of T3LHyp dehydratase and Pyr2C reductase with dual specificity between NADPH and NADH were found in cell-free extract prepared from A. brasilense cells grown not only on T3LHyp but also d-proline and d-lysine. Unexpectedly, although T4LHyp and C4DHyp also induced Pyr2C reductase, enzyme activity was clearly NADPH dependent. These results indicated that T3LHyp dehydratase and Pyr2C reductase are actually involved in the hypothetical T3LHyp pathway not only of mammalians but also bacteria, and that there are Pyr2C reductase isozymes with different inductivity by carbon sources and coenzyme specificity.


Identification and characterization of trans-3-hydroxy-l-proline dehydratase and Δ(1)-pyrroline-2-carboxylate reductase involved in trans-3-hydroxy-l-proline metabolism of bacteria.

Watanabe S, Tanimoto Y, Yamauchi S, Tozawa Y, Sawayama S, Watanabe Y - FEBS Open Bio (2014)

(A) Enzyme activities of cell-free extracts prepared from A. brasilense cells grown on several carbon sources. Values are the means ± SD, n = 2. Light- and dark-gray bars in Pyr2C reductase indicate NADPH- and NADH-dependent activities, respectively. (B) The transcriptional effect of carbon source on AbLhpI gene. Total RNAs (4 μg per lane) were isolated from the A. brasiliense cells grown on the indicated carbon sources.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3958920&req=5

f0010: (A) Enzyme activities of cell-free extracts prepared from A. brasilense cells grown on several carbon sources. Values are the means ± SD, n = 2. Light- and dark-gray bars in Pyr2C reductase indicate NADPH- and NADH-dependent activities, respectively. (B) The transcriptional effect of carbon source on AbLhpI gene. Total RNAs (4 μg per lane) were isolated from the A. brasiliense cells grown on the indicated carbon sources.
Mentions: First, we found that among three bacteria capable of metabolizing T4LHyp, only A. brasilense can grow on T3LHyp as a sole carbon source, not P. putida and P. aeruginosa; to our knowledge, this is the first report of T3LHyp metabolism by an organism(s) (Figs. 1E and S1). Next, we estimated whether the T4LHyp pathway is related to T3LHyp metabolism because of its structural similarity. However, all four enzymes involved in T4LHyp metabolism were induced only by T4LHyp (and C4DHyp), not by T3LHyp (Fig. 2A). On the other hand, significant activities of T3LHyp dehydratase and Pyr2C reductase with dual specificity between NADPH and NADH were found in cell-free extract prepared from A. brasilense cells grown not only on T3LHyp but also d-proline and d-lysine. Unexpectedly, although T4LHyp and C4DHyp also induced Pyr2C reductase, enzyme activity was clearly NADPH dependent. These results indicated that T3LHyp dehydratase and Pyr2C reductase are actually involved in the hypothetical T3LHyp pathway not only of mammalians but also bacteria, and that there are Pyr2C reductase isozymes with different inductivity by carbon sources and coenzyme specificity.

Bottom Line: In A. brasilense cells, T3LHyp dehydratase and NAD(P)H-dependent Δ(1)-pyrroline-2-carboxylate (Pyr2C) reductase activities were induced by T3LHyp (and d-proline and d-lysine) but not T4LHyp, and no effect of T3LHyp was observed on the expression of T4LHyp metabolizing enzymes: a hypothetical pathway of T3LHyp → Pyr2C → l-proline was proposed.On the other hand, Pyr2C reductase encoded to LhpI gene was a novel member of ornithine cyclodeaminase/μ-crystallin superfamily, differing from known bacterial protein.Furthermore, disruption of LhpI gene from A. brasilense led to loss of growth on T3LHyp, d-proline and d-lysine, indicating that this gene has dual metabolic functions as a reductase for Pyr2C and Δ(1)-piperidine-2-carboxylate in these pathways, and that the T3LHyp pathway is not linked to T4LHyp and l-proline metabolism.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama, Ehime 790-8566, Japan.

ABSTRACT
trans-4-Hydroxy-l-proline (T4LHyp) and trans-3-hydroxy-l-proline (T3LHyp) occur mainly in collagen. A few bacteria can convert T4LHyp to α-ketoglutarate, and we previously revealed a hypothetical pathway consisting of four enzymes at the molecular level (J Biol Chem (2007) 282, 6685-6695; J Biol Chem (2012) 287, 32674-32688). Here, we first found that Azospirillum brasilense has the ability to grow not only on T4LHyp but also T3LHyp as a sole carbon source. In A. brasilense cells, T3LHyp dehydratase and NAD(P)H-dependent Δ(1)-pyrroline-2-carboxylate (Pyr2C) reductase activities were induced by T3LHyp (and d-proline and d-lysine) but not T4LHyp, and no effect of T3LHyp was observed on the expression of T4LHyp metabolizing enzymes: a hypothetical pathway of T3LHyp → Pyr2C → l-proline was proposed. Bacterial T3LHyp dehydratase, encoded to LhpH gene, was homologous with the mammalian enzyme. On the other hand, Pyr2C reductase encoded to LhpI gene was a novel member of ornithine cyclodeaminase/μ-crystallin superfamily, differing from known bacterial protein. Furthermore, the LhpI enzymes of A. brasilense and another bacterium showed several different properties, including substrate and coenzyme specificities. T3LHyp was converted to proline by the purified LhpH and LhpI proteins. Furthermore, disruption of LhpI gene from A. brasilense led to loss of growth on T3LHyp, d-proline and d-lysine, indicating that this gene has dual metabolic functions as a reductase for Pyr2C and Δ(1)-piperidine-2-carboxylate in these pathways, and that the T3LHyp pathway is not linked to T4LHyp and l-proline metabolism.

No MeSH data available.