Seiya Watanabe

Identification and characterization of trans‐3‐hydroxy‐l‐proline dehydratase and Δ1‐pyrroline‐2‐carboxylate reductase involved in trans‐3‐hydroxy‐l‐proline metabolism of bacteria

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 Δ1‐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 Δ1‐piperidine‐2‐carboxylate in these pathways, and that the T3LHyp pathway is not linked to T4LHyp and l‐proline metabolism.

An enzymatic method to estimate the content of L-hydroxyproline

Post-translational hydroxylation of the L-proline residue mainly occurs in collagen; therefore, the L-hydroxyprolines (L-Hyp) synthesized, including trans-4-hydroxy-L-proline (T4LHyp) and trans-3-hydroxy-L-proline (T3LHyp), are important markers for directly measuring the content of collagen in several biological samples. The most frequently used method to estimate the content of L-Hyp is high-performance liquid chromatography (HPLC), which is inconvenient. In the present study, we attempted to estimate the content of L-Hyp using coupling systems with metabolic enzymes of the T4LHyp (hydroxyproline 2-epimerase (HypE) and cis-4-hydroxy-D-proline dehydrogenase (HypDH)) and T3LHyp pathways (T3LHyp dehydratase (T3LHypD) and Δ(1)-pyrroline-2-carboxylate reductase (Pyr2CR)) from microorganisms. We constructed a functional expression system of recombinant HypDH with a heterooligomeric structure in Escherichia coli cells. Enzymological characterization revealed that the β-subunit acted as a catalytic subunit, and also that assembly with other subunit(s) improved the kinetics for cis-4-hydroxy-D-proline and thermostability. By using a spectrophotometric assay with different wavelengths, the contents of T4LHyp and T3LHyp were successfully estimated within the ranges of 0.004-1mM and 0.05-1mM, respectively, and were consistent with those determined by HPLC. This enzymatic method was used to measure the content of T4LHyp in the acid-hydrolysate of collagen, and blood plasma.

Diversity in Guanosine 3,5-Bisdiphosphate (ppGpp) Sensitivity among Guanylate Kinases of Bacteria and Plants *

Background: The ppGpp signaling system is operative in plant chloroplasts and bacteria. Results: Chloroplast and cytosolic guanylate kinases (GKs) of plants are sensitive and insensitive to ppGpp, respectively, whereas bacterial GKs show diversity in ppGpp sensitivity. Conclusion: GTP biosynthesis in chloroplasts is controlled by ppGpp. Significance: Identification of the targets of ppGpp should provide insight into biological processes regulated by this nucleotide. The guanosine 3′,5′-bisdiphosphate (ppGpp) signaling system is shared by bacteria and plant chloroplasts, but its role in plants has remained unclear. Here we show that guanylate kinase (GK), a key enzyme in guanine nucleotide biosynthesis that catalyzes the conversion of GMP to GDP, is a target of regulation by ppGpp in chloroplasts of rice, pea, and Arabidopsis. Plants have two distinct types of GK that are localized to organelles (GKpm) or to the cytosol (GKc), with both enzymes being essential for growth and development. We found that the activity of rice GKpm in vitro was inhibited by ppGpp with a Ki of 2.8 μm relative to the substrate GMP, whereas the Km of this enzyme for GMP was 73 μm. The IC50 of ppGpp for GKpm was ∼10 μm. In contrast, the activity of rice GKc was insensitive to ppGpp, as was that of GK from bakers yeast, which is also a cytosolic enzyme. These observations suggest that ppGpp plays a pivotal role in the regulation of GTP biosynthesis in chloroplasts through specific inhibition of GKpm activity, with the regulation of GTP biosynthesis in chloroplasts thus being independent of that in the cytosol. We also found that GKs of Escherichia coli and Synechococcus elongatus PCC 7942 are insensitive to ppGpp, in contrast to the ppGpp sensitivity of the Bacillus subtilis enzyme. Our biochemical characterization of GK enzymes has thus revealed a novel target of ppGpp in chloroplasts and has uncovered diversity among bacterial GKs with regard to regulation by ppGpp.

Identification and Characterization of Bifunctional Proline Racemase/Hydroxyproline Epimerase from Archaea: Discrimination of Substrates and Molecular Evolution

Proline racemase (ProR) is a member of the pyridoxal 5’-phosphate-independent racemase family, and is involved in the Stickland reaction (fermentation) in certain clostridia as well as the mechanisms underlying the escape of parasites from host immunity in eukaryotic Trypanosoma. Hydroxyproline epimerase (HypE), which is in the same protein family as ProR, catalyzes the first step of the trans-4-hydroxy-L-proline metabolism of bacteria. Their substrate specificities were previously considered to be very strict, in spite of similarities in their structures and catalytic mechanisms, and no racemase/epimerase from the ProR superfamily has been found in archaea. We here characterized the ProR-like protein (OCC_00372) from the hyperthermophilic archaeon, Thermococcus litoralis (TlProR). This protein could reversibly catalyze not only the racemization of proline, but also the epimerization of 4-hydroxyproline and 3-hydroxyproline with similar kinetic constants. Among the four (putative) ligand binding sites, one amino acid substitution was detected between TlProR (tryptophan at the position of 241) and natural ProR (phenylalanine). The W241F mutant showed a significant preference for proline over hydroxyproline, suggesting that this (hydrophobic and bulky) tryptophan residue played an importance role in the recognition of hydroxyproline (more hydrophilic and bulky than proline), and substrate specificity for hydroxyproline was evolutionarily acquired separately between natural HypE and ProR.u3000A phylogenetic analysis indicated that such unique broad substrate specificity was derived from an ancestral enzyme of this superfamily.

Characterization of Flavin-Containing Opine Dehydrogenase from Bacteria.

Opines, in particular nopaline and octopine, are specific compounds found in crown gall tumor tissues induced by infections with Agrobacterium species, and are synthesized by well-studied NAD(P)H-dependent dehydrogenases (synthases), which catalyze the reductive condensation of α-ketoglutarate or pyruvate with L-arginine. The corresponding genes are transferred into plant cells via a tumor-inducing (Ti) plasmid. In addition to the reverse oxidative reaction(s), the genes noxB-noxA and ooxB-ooxA are considered to be involved in opine catabolism as (membrane-associated) oxidases; however, their properties have not yet been elucidated in detail due to the difficulties associated with purification (and preservation). We herein successfully expressed Nox/Oox-like genes from Pseudomonas putida in P. putida cells. The purified protein consisted of different α-, β-, and γ-subunits encoded by the OdhA, OdhB, and OdhC genes, which were arranged in tandem on the chromosome (OdhB-C-A), and exhibited dehydrogenase (but not oxidase) activity toward nopaline in the presence of artificial electron acceptors such as 2,6-dichloroindophenol. The enzyme contained FAD, FMN, and [2Fe-2S]-iron sulfur as prosthetic groups. On the other hand, the gene cluster from Bradyrhizobium japonicum consisted of OdhB 1 -C-A-B 2, from which two proteins, OdhAB1C and OdhAB2C, appeared through the assembly of each β-subunit together with common α- and γ-subunits. A poor phylogenetic relationship was detected between OdhB1 and OdhB2 in spite of them both functioning as octopine dehydrogenases, which provided clear evidence for the acquisition of novel functions by “subunit-exchange”. To the best of our knowledge, this is the first study to have examined flavin-containing opine dehydrogenase.

Functional characterization of aconitase X as a cis -3-hydroxy-L-proline dehydratase

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.

Ornithine cyclodeaminase/μ‐crystallin homolog from the hyperthermophilic archaeon Thermococcus litoralis functions as a novel Δ1‐pyrroline‐2‐carboxylate reductase involved in putative trans‐3‐hydroxy‐l‐proline metabolism

l‐Ornithine cyclodeaminase (OCD) is involved in l‐proline biosynthesis and catalyzes the unique deaminating cyclization of l‐ornithine to l‐proline via a Δ1‐pyrroline‐2‐carboxyrate (Pyr2C) intermediate. Although this pathway functions in only a few bacteria, many archaea possess OCD‐like genes (proteins), among which only AF1665 protein (gene) from Archaeoglobus fulgidus has been characterized as an NAD+‐dependent l‐alanine dehydrogenase (AfAlaDH). However, the physiological role of OCD‐like proteins from archaea has been unclear. Recently, we revealed that Pyr2C reductase, involved in trans‐3‐hydroxy‐l‐proline (T3LHyp) metabolism of bacteria, belongs to the OCD protein superfamily and catalyzes only the reduction of Pyr2C to l‐proline (no OCD activity) [FEBS Open Bio (2014) 4, 240–250]. In this study, based on bioinformatics analysis, we assumed that the OCD‐like gene from Thermococcus litoralis DSM 5473 is related to T3LHyp and/or proline metabolism (TlLhpI). Interestingly, TlLhpI showed three different enzymatic activities: AlaDH; N‐methyl‐l‐alanine dehydrogenase; Pyr2C reductase. Kinetic analysis suggested strongly that Pyr2C is the preferred substrate. In spite of their similar activity, TlLhpI had a poor phylogenetic relationship to the bacterial and mammalian reductases for Pyr2C and formed a close but distinct subfamily to AfAlaDH, indicating convergent evolution. Introduction of several specific amino acid residues for OCD and/or AfAlaDH by site‐directed mutagenesis had marked effects on both AlaDH and Pyr2C reductase activities. The OCC_00387 gene, clustered with the TlLhpI gene on the genome, encoded T3LHyp dehydratase, homologous to the bacterial and mammalian enzymes. To our knowledge, this is the first report of T3LHyp metabolism from archaea.

Characterization of iron-sulfur clusters in flavin-containing opine dehydrogenase

Flavin-containing opine dehydrogenase from Bradyrhizobium japonicum forms a heterooligomeric α4β4γ4 enzyme complex. An electron paramagnetic resonance spectroscopy analysis using wild-type and site-directed mutants revealed that [4Fe-4S] and [2Fe-2S] clusters bind to two different types of [Fe-S] binding sites in the γ- and α-subunits, respectively. The latter was found to be important for structural folding and enzyme catalysis.

Characterization of cis-4-hydroxy-D-proline dehydrogenase from Sinorhizobium meliloti

Abstract The hypO gene from Sinorhizobium meliloti, located within the trans-4-hydroxy-L-proline metabolic gene cluster, was first successfully expressed in the host Pseudomonas putida. Purified HypO protein functioned as a FAD-containing cis-4-hydroxy-D-proline dehydrogenase with a homomeric structure. In contrast to other known enzymes, significant activity for D-proline was found, confirming a previously proposed potential involvement in D-proline metabolism.

Characterization of a Novel cis-3-Hydroxy-L-proline Dehydratase and a trans-3-Hydroxy-L-proline Dehydratase from Bacteria

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).