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Identification of a human trans-3-hydroxy-L-proline dehydratase, the first characterized member of a novel family of proline racemase-like enzymes.

ABSTRACT: A family of eukaryotic proline racemase-like genes has recently been identified. Several members of this family have been well characterized and are known to catalyze the racemization of free proline or trans-4-hydroxyproline. However, the majority of eukaryotic proline racemase-like proteins, including a human protein called C14orf149, lack a specific cysteine residue that is known to be critical for racemase activity. Instead, these proteins invariably contain a threonine residue at this position. The function of these enzymes has remained unresolved until now. In this study, we demonstrate that three enzymes of this type, including human C14orf149, catalyze the dehydration of trans-3-hydroxy-L-proline to ?(1)-pyrroline-2-carboxylate (Pyr2C). These are the first enzymes of this subclass of proline racemase-like genes for which the enzymatic activity has been resolved. C14orf149 is also the first human enzyme that acts on trans-3-hydroxy-L-proline. Interestingly, a mutant enzyme in which the threonine in the active site is mutated back into cysteine regained 3-hydroxyproline epimerase activity. This result suggests that the enzymatic activity of these enzymes is dictated by a single residue. Presumably, human C14orf149 serves to degrade trans-3-hydroxy-L-proline from the diet and originating from the degradation of proteins that contain this amino acid, such as collagen IV, which is an important structural component of basement membrane.

SUBMITTER: Visser WF

PROVIDER: S-EPMC3381129 | biostudies-literature | 2012 Jun

REPOSITORIES: biostudies-literature

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Identification of a human trans-3-hydroxy-L-proline dehydratase, the first characterized member of a novel family of proline racemase-like enzymes.

Visser Wouter F WF Verhoeven-Duif Nanda M NM de Koning Tom J TJ

The Journal of biological chemistry 20120423 26

A family of eukaryotic proline racemase-like genes has recently been identified. Several members of this family have been well characterized and are known to catalyze the racemization of free proline or trans-4-hydroxyproline. However, the majority of eukaryotic proline racemase-like proteins, including a human protein called C14orf149, lack a specific cysteine residue that is known to be critical for racemase activity. Instead, these proteins invariably contain a threonine residue at this posit ...[more]

PMID: 22528483

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Project description: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 (AcnXType 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 (hypIS and hypIL, 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 (AcnXType IIa). A strain disrupted in the hypIS 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 Δ1-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 AcnXType 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).IMPORTANCE More than 50 years after the discovery of trans-4-hydroxy-l-proline (generally called 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).

Dataset Information

Identification of a human trans-3-hydroxy-L-proline dehydratase, the first characterized member of a novel family of proline racemase-like enzymes.

Publications

Identification of a human trans-3-hydroxy-L-proline dehydratase, the first characterized member of a novel family of proline racemase-like enzymes.

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