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

Watanabe, Seiya; Tajima, Kunihiko; Fujii, Satoshi; Fukumori, Fumiyasu; Hara, Ryotaro; Fukuda, Rio; Miyazaki, Mao; Kino, Kuniki; Watanabe, Yasuo

doi:10.1038/srep38720

Download PDF

Article
Open access
Published: 08 December 2016

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

Seiya Watanabe^1,2,3,
Kunihiko Tajima⁴,
Satoshi Fujii⁵,
Fumiyasu Fukumori⁶,
Ryotaro Hara⁷,
Rio Fukuda²,
Mao Miyazaki²,
Kuniki Kino^7,8 &
…
Yasuo Watanabe^1,2

Scientific Reports volume 6, Article number: 38720 (2016) Cite this article

2269 Accesses
5 Citations
Metrics details

Subjects

Abstract

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.

Crystal structures of aconitase X enzymes from bacteria and archaea provide insights into the molecular evolution of the aconitase superfamily

Article Open access 07 June 2021

An iron (II) dependent oxygenase performs the last missing step of plant lysine catabolism

Article Open access 10 June 2020

Structures of fungal and plant acetohydroxyacid synthases

Article 08 July 2020

Introduction

L-Hydroxyproline has been found in certain proteins, particularly in collagen, and in some peptide antibiotics. In mammalian systems, the L-proline residue is post-translationally hydroxylated to trans-4-hydroxy-L-proline [(2S,4R)-4-hydroxyproline] or trans-3-hydroxy-L-proline [(2S,3S)-3-hydroxyproline] by a diverse set of prolyl 4-hydroxylase (EC 1.14.11.2) and prolyl 3-hydroxylase (EC 1.14.11.7) with different protein substrates, respectively¹. A number of bacterial and fungal enzymes are known to directly hydroxylate free L-proline to trans-4-hydroxy-L-proline², trans-3-hydroxy-L-proline^3,4, cis-4-hydroxy-L-proline [(2S,4S)-4-hydroxyproline]⁵, or cis-3-hydroxy-L-proline [(2S,3R)-3-hydroxyproline]⁶, which are also found as components of several peptide antibiotics produced by these microorganisms^7,8,9,10,11.

Among the several stereoisomers of L-hydroxyproline, trans-4-hydroxy-L-proline is the most common in nature, and is converted to pyruvate and glyoxylate via three intermediates in mammals (Table S1)¹. On the other hand, a number of bacteria, including Pseudomonas aeruginosa PAO1 and Pseudomonas putida KT2440, metabolize trans-4-hydroxy-L-proline to α-ketoglutarate by four enzymes (encoded by LhpA~F genes) (Fig. 1a and Table S1)^12,13. In contrast to trans-4-hydroxy-L-proline, the metabolic pathways of trans-3-hydroxy-L-proline in bacteria and mammalians may be similar, and consist of a dehydratase (LhpG) and a reductase (LhpH or LhpK), by which trans-3-hydroxy-L-proline is converted to L-proline via Δ¹-pyrroline-2-carboxylate (Fig. 1b and Table S1)^14,15. It is likely that the reductase is also involved in cis-3-hydroxy-L-proline metabolism in bacteria, in which Δ¹-pyrroline-2-carboxylate is produced by a different dehydratase than the LhpH protein (LhpJ) (Fig. 1c and Table S1)¹⁶. These LhpA~K genes often cluster together with the putative L-hydroxyproline (ABC-type) transporter genes, transcriptional regulator gene, aconitase-like gene (LhpI) and proline racemase-like gene (LhpL) on bacterial genomes (Fig. 1d; referred to as the “L-hydroxyproline gene cluster”)^12,13,15,16. Among these hypothetical genes, it is likely that the LhpI gene (protein) in particular is not necessary for the known pathway of L-hydroxyproline metabolism due to its putative function, based on amino acid sequence similarity.

The aconitase superfamily contains four functional hydro-lyase enzymes: aconitase (EC 4.2.1.3; Acn), 2-methylcitrate dehydratase (EC 4.2.1.79; AcnD), homoaconitase (EC 4.2.1.114; HACN), and isopropylmalate isomerase (EC 4.2.1.33; IPMI), which have been classified into eight phylogenetic subfamilies: (1) AcnA of bacteria¹⁷; (2) AcnB of bacteria¹⁸; (3) mitochondrial Acn (mAcn)¹⁹; (4) cytoplasmic Acn and iron regulatory protein (IRP) of mammalians²⁰; (5) AcnD of bacteria²¹; (6) HACN of bacteria and archaea^22,23; (7) IPMI of bacteria, archaea, and fungi^24,25,26; (8) function unknown aconitase X (AcnX) (Fig. 2). All of these subfamilies (referred to as “Acn enzymes”), except for the last one, catalyze the homologous stereospecific isomerization of α- to β-hydroxyl acids by sequential dehydration and hydration (anti-elimination/addition), and their active centers contain a [4Fe-4S] iron-sulfur cluster. By contrast, AcnX (subfamily) was initially discovered by a comparative analysis of archaeal genomes in 2003²⁷. Although the secondary structural elements of Acn enzymes may also be conserved in this subfamily, the sequence similarities between them are insignificant. The AcnX subfamily has been further classified into “AcnX_{Type I}”, consisting of a single polypeptide from bacteria, and “AcnX_{Type II}” found in a number of bacteria (AcnX_{Type IIa}) and archaea (AcnX_{Type IIb}), which consists of (fragmented) small and large polypeptide chains. The small polypeptide (subunit) of AcnX_{Type II} is homologous to the so-called “fourth domain” of Acn enzymes as well as the IPMI and HACN of bacteria and archaea (Fig. 2)^22,23,24,25. Among the three AcnX subfamilies, LhpI protein corresponds to AcnX_{Type I}.

We report here that LhpI protein catalyzes the dehydration of cis-3-hydroxy-L-proline to Δ¹-pyrroline-2-carboxylate, by a reaction that was not homologous with those of known Acn enzymes, and contains mononuclear Fe(III), but not the [4Fe-4S] cluster. Bacterial growth and transcriptional analysis suggested that this enzyme is surely involved in cis-3-hydroxy-L-proline metabolism.

Results and Discussion

Identification of substrates and functions

A genetic analysis of the L-hydroxyproline gene cluster (PA1252-1269) from Pseudomonas aeruginosa PAO1 was recently reported in detail (Fig. 1d)²⁸. Among the 18 components, 10 genes including PA1259 (LhpI) were significantly induced by trans-4-hydroxy-L-proline. Furthermore, the LhpI mutant, constructed by transposon insertion, as well as mutants of the known trans-4-hydroxy-L-proline metabolic gene lost the ability to grow on trans-4-hydroxy-L-proline. However, this phenotype may be due to a polar effect on downstream genes²⁸. The non-polar LhpI deletion mutant (SMb20269; Fig. 1d) of Sinorhizobium meliloti 1021 was previously reported to grow normally on trans-4-hydroxy-L-proline²⁹. Therefore, there has been no evidence to show that the LhpI gene plays a role in the metabolism of L-hydroxyproline.

The recombinant (His)₆-tagged PaLhpI protein was successfully expressed in P. putida cells, and purified to homogeneity using a nickel-chelating affinity column (Fig. 3a,b). The apparent molecular masses, estimated by SDS-PAGE and analytic gel filtration, were 60 and 67 kDa, indicating a monomeric structure. Initially, no hydro-lyase activity toward citrate and/or cis-acotinate was observed in this protein. Therefore, in the next approach for functional analysis, eight proline derivatives (10 mM; Fig. S1) were tested as substrates in Tris-HCl buffer (pH 8.0) without additives. Among them, only cis-3-hydroxy-L-proline was consumed in a time-dependent manner (specific activity of 39.5 μmol·min⁻¹·mg⁻¹) (Fig. 4a), and the reaction product was identified as Δ¹-pyrroline-2-carboxylate by ¹H NMR (Fig. 4g). This result confirmed that when the reaction was performed in the co-presence of Δ¹-pyrroline-2-carboxylate reductase¹⁵, L-proline was produced in a time-dependent manner (Fig. 4b). Collectively, these results suggest that the PaLhpI protein catalyzes (only) the irreversible dehydration reaction of cis-3-hydroxy-L-proline to Δ¹-pyrroline-2-carboxylate via a putative Δ²-pyrroline-2-carboxylate intermediate (Fig. 1c). Since cis-3-hydroxy-L-proline possesses the hydroxyl group and proton to be eliminated at the same “anti” positions as Acn enzymes, it is likely that trans-3-hydroxy-L-proline (“syn” positions) is not a substrate for this protein.

In order to investigate substrate specificity in more detail, we prepared a unique 2,3-cis-3,4-cis-3,4-dihydroxy-L-proline [(2 S,3 S,4 R)-3,4-dihydroxyproline] from cis-3-hydroxy-L-proline by L-proline cis-4-hydroxylase from S. meliloti³⁰. This proline derivative was an additional substrate for PaLhpI, whereas specific activity (0.130 μmol·min⁻¹·mg⁻¹), estimated by HPLC analysis, was ~300-fold lower than that of cis-3-hydroxy-L-proline (Fig. 4c). Although the ¹H NMR analysis did not identify the enzyme product directly (Fig. S2), cis-4-hydroxy-L-proline was produced by coupling with Δ¹-pyrroline-2-carboxylate reductase (Fig. 4d); the reaction product of 2,3-cis-3,4-cis-3,4-dihydroxy-L-proline must be a Δ¹-pyrroline-4S-hydroxy-2-carboxylate. Furthermore, no dehydration (degradation) was observed in L-serine, in spite of carbons 1~3 as well as the hydroxyl and amino groups being identical to those of cis-3-hydroxy-L-proline (Fig. S3). These results indicate that PaLhpI recognizes not only the framework and chiral selectivity of 3-hydroxyproline, but also the 4′-functional group of cis-3-hydroxy-L-proline.

Labrenzia aggregata IAM 12614 possesses the L-hydroxyproline gene cluster, which contains the LhpJ gene (SIAM614_RS19325) instead of the LhpI gene (Fig. 1d)¹⁶. The LhpJ protein belongs to the muconate lactonizing enzyme subclass of the enolase superfamily, distinct from the Acn superfamily, and shows the bifunctional activity of (1) the reversible 2-epimerization of cis-3-hydroxy-L-proline to trans-3-hydroxy-D-proline and (2) the irreversible dehydration of cis-3-hydroxy-L-proline to Δ¹-pyrroline-2-carboxylate (Fig. 1c); the latter is homologous with the activities of hydroxyproline betaine 2-epimerase³¹ and proline betaine racemase³², other members in this protein superfamily. On the other hand, epimerization activity was not found in PaLhpI (Fig. 4g), possibly due to the different catalytic mechanisms of the proteins, as described below.

Catalytic properties

In the absence of substrate under oxidative conditions, one of the iron ions, so-called “Fe_α” (Fig. 5a), is easily lost from the [4Fe-4S] cluster in Acn enzymes, leading to the catalytically inactive [3Fe-4S] state; therefore, reconstruction (reactivation) is typically necessary under reducing conditions³³. Purified PaLhpI was brown in color, indicating the presence of cofactor(s) (Fig. 3a), whereas inactivation was not detected after three months at −35 °C. The pH dependence of dehydration activity with cis-3-hydroxy-L-proline was estimated by a colorimetric method based on the reaction of 2-aminobenzaldehyde with Δ¹-pyrroline-2-carboxylate^14,15: optimum pH of 8.0–9.5 (Fig. 3c). Moreover, we developed a more conventional spectrophotometric assay method using NADPH-dependent Δ¹-pyrroline-2-carboxylate reductase as a coupling enzyme (see Fig. 4b). Kinetic parameters with cis-3-hydroxy-L-proline are shown in Fig. 4h. The specific activity value, 41.3 μmol·min⁻¹·mg⁻¹, was similar to that obtained by HPLC.

Among the various divalent metal ions tested, only Zn²⁺, Cd²⁺, and Hg²⁺ inhibited activity (Fig. S4a), and their IC₅₀ values were 9.75 × 10⁻³, 2.05 × 10⁻³, and 2.47 × 10⁻² mM, respectively (Fig. 6a). The pattern was competitive, and the K_i value for Zn²⁺ was 1.07 × 10⁻³ mM (Fig. S4b). In the case of Acn enzymes, only mitochondrial Acn from mammals was inhibited by Zn²⁺ (K_i = 2.00 × 10⁻³ mM)³⁴. Furthermore, pyrrole-2-carboxylate (and its derivative 2-thiophenecarboxylate) inhibited the activity of PaLhpI (Fig. 6a), whereas several characteristic inhibitors for Acn enzymes including trans-aconitate, fluorocitrate, and oxalomalate did not. Pyrrole-2-carboxylate is structurally analogous to the (putative) transition state of cis-3-hydroxy-L-proline, Δ²-pyrroline-2-carboxylate (see Fig. 1c), and its IC₅₀ value is markedly higher than those of divalent metal ions: 440-fold that of Cd²⁺. Collectively, these results strongly indicate that the substrate binding pocket of the PaLhpI protein is not similar to Acn enzymes, and that binding sites for divalent metal ions and pyrrole-2-carboxylate are spatially distinct.

Characterization of other AcnX_{Type I} enzymes

Although the bacterial LhpI gene is often located within the L-hydroxyproline gene cluster, there are several combinations of trans-4-hydroxy-L-proline and/or trans-3-hydroxy-L-proline metabolic genes. For example, there are two types of cis-4-hydroxy-D-proline dehydrogenases¹³: P. aeruginosa PAO1 etc., α₄β₄γ₄-type enzyme encoded by LhpB (encoding to β-subunit), LhpC (α-subunit) and LhpD genes (γ-subunit); Agrobacterium tumefaciens C58 etc., homomeric-type enzyme encoded by LhpB gene (Fig. 1d). Furthermore, there is no sequence similarity between LhpH and LhpK proteins encoding to Δ¹-pyrroline-2-carboxylate reductase. Although the AcnX protein was originally considered to only be present in archaea and bacteria²⁷, a homology search using the Protein-BLAST program revealed that a large number of fungi possess the LhpI homologous gene; the ANI_1_578044 gene from Aspergillus niger CBS 513.88 is closely located to the putative trans-3-hydroxy-L-proline dehydratase gene (Fig. 1d)¹⁴.

Therefore, in order to elucidate the catalytic functions of other AcnX_{Type I} proteins, we enzymatically investigated Atu4683 from A. tumefaciens C58 (AtLhpI) and TRIREDRAFT_59073 from Trichoderma reesei QM6a (TrLhpI), and found 46.2% and 51.7% sequence identity with PaLhpI, respectively. The (His)₆-tagged AtLhpI and TrLhpI proteins were expressed in Escherichia coli, purified, and characterized (Fig. 3a). As observed for PaLhpI, among eight proline derivatives (Fig. S1), only cis-3-hydroxy-L-proline was consumed in a time-dependent manner by both proteins, and similar inhibition by Zn²⁺ was observed (Fig. 6b,c). By contrast, the k_cat/K_m value of TrLhpI was 316- and 83.4-fold lower than those of PaLhpI and AtLhpI, respectively (Fig. 4h). Although analysis by an amino acid analyzer suggested the reaction product of TrLhpI is Δ¹-pyrroline-2-carboxylate (specific activity of 3.75 μmol·min⁻¹·mg⁻¹), the consumption rate of cis-3-hydroxy-L-proline by coupling assay with Δ¹-pyrroline-2-carboxylate reductase decreased significantly (Fig. 4e,f). Furthermore, no effect of pyrrole-2-carboxylate (and 2-thiophenecarboxylate) was found on the activity (Fig. 6c). These results suggest that the fungal TrLhpI protein may well have an entirely different main (physiological) substrate from cis-3-hydroxy-L-proline.

Putative active sites

There are ~23 amino acid residues that are completely (or highly) conserved between Acn enzymes (yellow- and orange-shadowed letters in Fig. 7), implying similar structural organization and catalytic mechanisms³⁵. However, most of these residues had no readily detectable counterpart in AcnX_{Type I} proteins. On the other hand, several amino acid residues were completely (or highly) conserved within the AcnX subfamily only (Fig. 7 and Fig. S5), and some of them may be located around the active sites of Acn enzymes. Based on these insights, we selected thirty amino acid residues at sites 1~4, 9, 11~14, 17, and 21~23 for a site-directed mutagenic study, and constructed each alanine mutant of PaLhpI (Fig. 5b). Among them, only D35A (136%), C207A (80.5%), and N457A (55.9%) mutants showed similar activity to the wild-type (WT) enzyme, whereas C275A, W292A, H404A, C516A, and Y542A mutants were not expressed in host cells, S66A, S70A, E294A, S295A, S303A, T309A, C518A, and K538A mutants were inactive, and C68A (0.4%), H459A (0.4%), D523A (14.4%) and S545A (7.3%) mutants decreased the activity significantly (the values in the parentheses are the expression of specific activity relative to the WT enzyme), suggesting their potential role(s) in catalysis and/or structure folding. Among them, Ser66 at site 2 may play a role in abstracting a proton from the Cα of cis-3-hydroxy-L-proline as a catalytic base, as in Acn enzymes³³. However, in contrast to Cys275 of PaLhpI, mutation of histidine of Acn enzymes at site 8 does not markedly change activity levels from those of the WT enzyme³⁶, probably caused by the lack of a direct ligand for [4Fe-4S] cluster, as described below.

Putative iron binding sites

Inhibition by the Hg²⁺ and Cd²⁺ of LhpI protein(s) is observed in many proteins that possess a cysteine residue(s) as the active site: furthermore, an incubation with Hg²⁺ results in irreversible inactivation (Fig. S4c). In the case of Acn enzymes, three iron ions of the [4Fe-4S] cluster were coordinated by three conserved cysteine residues (orange-shadowed letters at sites 15, 18, and 20 in Fig. 5a and Fig. 7)³³, among which PaLhpI possessed only one equivalent residue to site 18 (Cys516). Therefore, as other potential ligand(s), we selected Cys207, Cys275, Glu294, Asn457, and Cys518 at sites 5, 8, 10, 15, and 19, the corresponding residues of the Acn enzymes that were located around the [4Fe-4S] cluster, and measured the iron content in each alanine mutant by a colorimetric assay. As results, WT, C207A, E294A, N457A, and C518A contained 0.84, 0.76, 0.01>, 1.63, and 0.52 iron equivalents per (monomeric) protein (Fig. 5b): Cys275A and C516A mutants were not expressed in host cells, as described above. These findings indicate that PaLhpI contains an iron ion (but not [4Fe-4S] cluster) that is necessary for catalysis, and that Cys275, Glu294, and Cys516 at sites 8, 10, and 18 may be important for substrate binding (pink-shadowed letters). Complete conservation at these sites in the AcnX subfamily appears to support this hypothesis (Fig. S5). It is noteworthy that in the catalytic mechanism of the enolase type of cis-3-hydroxy-L-proline dehydratase (LhpJ), an enolate anion intermediate is stabilized by an essential Mg²⁺ ligated by two tightly conserved aspartate residues and one glutamate residue, and two lysine residues function as a basic and/or acidic catalyst¹⁶. Therefore, although AcnX_{Type I} has no evolutionary relationship with the enolase type enzyme, the catalytic mechanism including metal ion binding may be more similar than those of Acn enzymes.

Non-heme iron analysis

In order to examine the nature of non-heme iron in more detail, electron paramagnetic resonance (EPR) analysis was performed using AtLhpI. AtLhpI treated with dithionite (Na₂S₂O₄) exhibited EPR signals at g = 5.40, 4.65, and 2.00 (Fig. 5c), whereas reduced rubredoxin, a typical mononuclear iron-sulfur protein, was EPR-silent. This EPR spectrum is markedly different from those of other reduced iron-sulfur clusters, such as [2Fe-2S] ferredoxin, the [2Fe-2S] Rieske center, [3Fe-4S] ferredoxin, [3Fe-4S] Acn enzymes, and [4Fe-4S] ferredoxin, the EPR signals of which were observed in the g ≈ 2 region³⁷. Furthermore, it is interesting to note that the observed g-values of AtLhp1 were not a typical high-spin (S = 5/2) electronic ground state. Similar EPR spectra have been reported for cytochrome c’ from some photosynthetic bacteria, the electronic ground states of which are interpreted as quantum mechanical admixtures of an intermediate-spin state (S = 3/2) and high-spin state (S = 5/2)^38,39. The S = 3/2 and S = 5/2 components at the ground state of the iron center in AtLhp1 are estimated to be 49 and 51%, respectively, from observed g-values (5.40 and 4.65). Although the electronic ground state of iron is still ambiguous, these results suggest that the iron site in AtLhpI is an unprecedented mononuclear Fe(III) center that has a low redox potential that is resistant to the reduction of dithionite, but not an iron-sulfur cluster. In rubredoxin, Fe(III) is coordinated to four cysteinyl sulfurs occurring on two Cys-X₂-Cys segments or one Cys-X₂-Cys and one Cys-Cys⁴⁰. By contrast, in order to coordinate the Fe(II) center, “cupin superfamily” proteins have a conserved motif, in which no cysteine is contained as a ligand: His-X₂-[His/Asp]-X₄-Glu-X_n-His⁴¹. These specific motifs are not found in the sequences of AcnX enzymes (Fig. S5).

AcnX_{Type IIb} proteins possess two homologous cysteine residues, not only at site 18 but also site 15, with Acn enzymes (green-shadowed cysteine in Fig. 7 and Fig. S5). On the other hand, in the HACN subfamily, only enzymes from methanogenic archaea, including Methanococcus jannaschii, have the “fourth cysteine” at site 7 (light-green shadowed cysteine), which may be located near a sulfur atom in the [4Fe-4S] cluster; however, its role is currently unclear (Fig. 5a)^42,43. Indeed, we have already found that purified AcnX_{Type IIb} protein (from Aeropyrum pernix K1) also contains iron ion(s), although functional annotation is in progress (unpublished). Therefore, the EPR analysis of AcnX_{Type IIb} protein would be useful for further understanding of the unique binding mode of Fe(III) of AcnX subfamily, and identifying which of the [4Fe-4S] cluster, or Fe(III), common ancestors of the Acn superfamily possessed.

Degradation pathway of L-hydroxyproline(s) in P. aeruginosa PAO1

To the best of our knowledge, cis-3-hydroxy-L-proline has only been found as one of the building blocks of telomycin, a peptide antibiotic produced by Streptomyces canus C159^7,8, and these bacteria may also hydroxylate free L-proline to cis-3-hydroxy-L-proline enzymatically⁶. Since the amount of cis-3-hydroxy-L-proline in nature is markedly less than that of trans-4-hydroxy-L-proline, the possibility of catabolism by (micro)organisms had not previously been considered. In order to estimate the potential metabolism of L-hydroxyproline, P. aeruginosa PAO1 was cultivated in minimal medium supplemented with L-proline, trans-4-hydroxy-L-proline, trans-3-hydroxy-L-proline, or cis-3-hydroxy-L-proline as the sole carbon source. Measurements of concentration by an amino acid analyzer revealed that cis-3-hydroxy-L-proline (but not trans-3-hydroxy-L-proline) was consumed in a time-dependent manner: 72% degradation after 20 days (Fig. 8a,b). Furthermore, in comparisons with L-proline in a quantitative real-time PCR (qRT-PCR) analysis, the PaLhpI gene was induced not only by trans-4-hydroxy-L-proline and cis-4-hydroxy-D-proline, but also by cis-3-hydroxy-L-proline, whereas the PaLhpA and PaLhpH genes were only induced by trans-4-hydroxy-L-proline and cis-4-hydroxy-D-proline (Fig. 8e). Since the co-presence of trans-4-hydroxy-L-proline clearly enhanced the metabolism of cis-3-hydroxy-L-proline (Fig. 8c), slow degradation may be due to the lack of significant induction of the PaLhpH gene. Similarly, Starkeya novella DSM 506, which possesses the LhpJ gene (Fig. 1d), may utilize cis-3-hydroxy-L-proline as a carbon source, and transcription is up-regulated by cis-3-hydroxy-L-proline (and trans-4-hydroxy-L-proline)¹⁶. Although the co-presence of trans-4-hydroxy-L-proline also resulted in the degradation of trans-3-hydroxy-L-proline, half of the trans-3-hydroxy-L-proline consumed accumulated as a cis-3-hydroxy-D-proline (Fig. 8d), indicating the presence of trans-3-hydroxy-L-proline metabolic enzyme(s). We herein demonstrated that PaLhpL gene (PA1255; Fig. 1d) in the L-hydroxyproline gene cluster encodes a bifunctional dehydratase and 2-epimerase toward trans-3-hydroxy-L-proline as the leading candidate (Supplementary Discussion and Fig. S6).

Overall, the L-hydroxyproline gene cluster from microorganisms is often related not only to the metabolism of trans-4-hydroxy-L-proline, but also to that of cis-3-hydroxy-L-proline and trans-3-hydroxy-L-proline, suggesting that the origin of these free L-hydroxyproline compounds in nature is the degradation of several peptide antibiotics, such as telomycin (containing cis-3-hydroxy-L-proline and trans-3-hydroxy-L-proline)^7,8, microcolin A (cis-4-hydroxy-L-proline)⁹, pneumocandins (trans-4-hydroxy-L-proline and trans-3-hydroxy-L-proline)¹⁰, and etamycin (cis-4-hydroxy-D-proline)¹¹, rather than collagen.

Conclusion

We herein showed that LhpI protein catalyzes the dehydration of cis-3-hydroxy-L-proline to Δ¹-pyrroline-2-carboxylate: over 10 years after the discovery of AcnX (subfamily) in Acn superfamily²⁷, the functional characterization can now be done successfully. The gene context in the bacterial genome was of considerable help in estimating the potential substrate, rather than the amino acid sequence similarity. Although cis-3-hydroxy-L-proline is believed to hardly exist in nature, our findings may indicate the significant physiological role in (micro)organisms, similar to trans-3-hydroxy-L-proline^14,15. Until now, the Acn superfamily (enzymes) was a typical example suitable for a recruitment hypothesis of enzyme evolution proposed by Jensen (1976)⁴⁴; gene duplication of multi-specific enzymes containing a [4Fe-4S] cluster, followed by narrowing of substrate specificity. Moreover, this functional annotation of a Fe(III)-containing AcnX protein must promote interest in common ancestors of the Acn superfamily, including the nature of the iron-sulfur cluster.

Methods

Plasmid construction for the expression of recombinant proteins

The primer sequences used in this study are shown in Table S2. PaLhpI, TrLhpI, and AtLhpI genes were amplified by PCR using primers containing appropriate restriction enzyme sites at the 5′- and 3′-ends and the genome DNA of P. aeruginosa PAO1, T. reesei QM6a, or A. tumefaciens C58 as a template. Each amplified DNA fragment was introduced into BamHI-HindIII sites in pQE-80L (Qiagen), a plasmid vector for conferring an N-terminal (His)₆ tag on the proteins expressed, in order to obtain pQE/PaLhpI, pQE/TrLhpI, and pQE/AtLhpI, respectively. Regarding the expression of the PaLhpI gene in P. putida cells, a DNA fragment of the (His)₆-PaLhpI-t₀ terminator was amplified by PCR using pQE/PaLhpI as a template, and introduced into the SalI-EcoRI sites in pUCP26KmAhpC_p⁴⁵ in order to obtain pUCP/PaLhpI. A site-directed mutation was introduced into the PaLhpI gene by sequential steps of PCR using sense and antisense primers and pUCP/PaLhpI as a template.

Expression and purification of the recombinant protein

P. putida KT2442-oxyR1⁴⁶ harboring pUCP/PaLhpI was grown at 30 °C overnight in LB medium containing kanamycin (50 mg/liter), whereas E. coli strain DH5α harboring pQE/TrLhpI or pQE/AtLhpI was grown at 37 °C to a turbidity of 0.6 at 600 nm in Super broth medium containing ampicillin (50 mg/liter). After the addition of 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG), the culture was grown at 18 °C for a further 18 h in order to induce the expression of the (His)₆-tagged protein. Grown cells were harvested, suspended in Buffer A (50 mM sodium phosphate buffer (pH 8.0) containing 300 mM NaCl and 10 mM imidazole), disrupted by sonication, and then centrifuged. The supernatant was loaded onto a Ni-NTA Superflow column (Qiagen) equilibrated with Buffer A linked to the BioAssist eZ system (TOSOH). The column was washed with Buffer B (50 mM sodium phosphate buffer (pH 8.0) containing 300 mM NaCl, 10% (v/v) glycerol, and 50 mM imidazole). The enzymes were then eluted with Buffer C (pH 8.0, as Buffer B but containing 250 mM instead of 50 mM imidazole), concentrated by ultrafiltration, dialyzed against 50 mM Tris-HCl buffer (pH 8.0) containing 50% (v/v) glycerol, and stored at –35 °C until use. The native molecular mass of the recombinant protein was estimated by gel filtration using a HiLoad 16/60 Superdex 200 pg column (GE Healthcare) equilibrated with 50 mM Tris-HCl buffer (pH 8.0). A high-molecular-weight gel filtration calibration kit (GE Healthcare) was used for molecular markers. Protein concentrations were measured by the method of Lowry et al.⁴⁷ with bovine serum albumin as the standard.

Screening of the proline derivative library for the LhpI protein

The purified PaLhpI protein (5 μg) was added to 50 mM Tris-HCl buffer (pH 8.0) (1 ml) containing 10 mM substrate (Fig. S1), and incubated at 30 °C. After varying the incubation times, the enzyme reaction was stopped by briefly incubating at −80 °C. Substrate consumption was estimated by HPLC using an Ultron ES-CD chiral separation column (150 × 2.0 mm, Shinwa Chemical Industries)⁴⁸ and/or Hitachi L-8900 PH Amino Acid Analyzer (Hitachi, Tokyo, Japan).

Enzyme assay

Unless noted otherwise, cis-3-hydroxy-L-proline dehydratase was assayed spectrophotometrically in the coupling system with Δ¹-pyrroline-2-carboxylate reductase (LhpH protein from P. aeruginosa PAO1)¹⁵. The reaction mixture consisted of 50 mM Tris-HCl (pH 8.0), 0.15 mM NADPH, and purified Δ¹-pyrroline-2-carboxylate reductase (10 μg). The reaction was initiated by the addition of 100 mM cis-3-hydroxy-L-proline (100 μl) with a final reaction volume of 1 ml. One unit of enzyme activity refers to 1 μmol NADPH consumed/min. K_m and k_cat values were calculated by a Lineweaver-Burk plot. The enzyme was alternatively assayed by a colorimetric method based on the reaction of 2-aminobenzaldehyde with Δ¹-pyrroline-2-carboxylate, which yields a yellow reaction product^13,14. This method was used to clarify the optimum pH for enzyme activity. Potential Acn activity in PaLhpI protein was measured spectrophotometrically by monitoring the change of absorption at 240 nm derived from unsaturated cis-aconitate⁴⁹.

Identification of reaction products

In order to remove glycerol in the storage buffer, purified PaLhpI was dialyzed at 4 °C overnight in 50 mM K₂HPO₄/KH₂PO₄ buffer (pH 7.0), lyophilized, and solvated in D₂O (600 μl) containing 20 mM cis-3-hydroxy-L-proline or 2,3-cis-3,4-cis-3,4-dihydroxy-L-proline. NMR spectra were recorded at 25 °C on a JEOL JNM-EC400 NMR spectrometer (JEOL Ltd., Tokyo, Japan) operating at 400 MHz. 2,2-Dimethyl-2-silapentane-5-sulfonate was used as an internal standard. 2,3-cis-3,4-cis-3,4-Dihydroxy-L-proline was enzymatically synthesized from cis-3-hydroxy-L-proline by L-proline cis-4-hydroxylase from S. meliloti, as described previously³⁰.

Non-heme iron analysis

The content of non-heme iron was assessed using a Metallo Assay kit (AKJ Global Technology, Japan). The (potential) iron-sulfur cluster was analyzed by electron paramagnetic resonance (EPR) using a JEOL TE-300 X-band spectrometer operating with a 100-kHz field modulation. A temperature-dependent analysis was performed in the range of 10 to 40 K using a LTR-3 liquid helium cryostat (Air Products). The purified AtLhpI protein (~40 mg/ml) was prepared in 50 mM Tris-HCl (pH8.0) containing 50% (v/v) glycerol for this purpose, and treated with a 10-fold excess amount of Na₂S₂O₄ under a nitrogen atmosphere. EPR spectra were recorded using the following representative conditions; microwave frequency; 8.9995 GHz, microwave power; 5.0 mW, 100 kHz modulation magnitude; 0.63 mT, center field; 280 ± 250 mT, sweep time; 4.0 min, time constant; 0.1 sec, and receiver amplitude; 500. In the present study, g-values were evaluated based on the g-value of the Li-salt of tetracyanoquinodimethane (2.0025) as an external standard. The magnetic field strength of EPR spectra was calibrated using the hyperfine coupling constant (8.69 mT) of the Mn(II) ion doped in MgO powder.

Growth and gene expression analysis of P. aeruginosa PAO1

P. aeruginosa PAO1 was cultured aerobically with vigorous shaking at 30 °C in 5 ml of minimal medium¹⁵ supplemented with a 30 mM carbon source(s). The concentration of L-hydroxyproline in medium after varying cultivation times was analyzed using an amino acid analyzer (L-8900; Hitachi, Tokyo, Japan). The preparation of RNA samples and one-step real-time RT-PCR were performed as described previously⁵⁰. The primers used for RT-PCR are listed in Table S2. The PA0393 gene encoding pyrroline-5-carboxylate reductase was used as an internal control.

Sequence comparison

Protein sequences were analyzed using the Protein-BLAST and Clustal W programs distributed by DDBJ (DNA Data Bank of Japan).

Additional Information

How to cite this article: Watanabe, S. et al. Functional characterization of aconitase X as a cis-3-hydroxy-L-proline dehydratase. Sci. Rep. 6, 38720; doi: 10.1038/srep38720 (2016).

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

Adams, E. & Frank, L. Metabolism of proline and the hydroxyprolines. Annu. Rev. Biochem. 49, 1005–1061 (1980).
CAS PubMed Google Scholar
Shibasaki, T., Mori, H., Chiba, S. & Ozaki, A. Microbial proline 4-hydroxylase screening and gene cloning. Appl. Environ. Microbiol. 65, 4028–4031 (1999).
CAS PubMed PubMed Central Google Scholar
Petersen, L., Olewinski, R., Salmon, P. & Connors, N. Novel proline hydroxylase activities in the pneumocandin-producing fungus Glarea lozoyensis responsible for the formation of trans 3- and trans 4-hydroxyproline. Appl. Microbiol. Biotechnol. 62, 263–267 (2003).
CAS PubMed Google Scholar
Houwaart, S., Youssar, L. & Hüttel, W. Pneumocandin biosynthesis: involvement of a trans-selective proline hydroxylase. Chembiochem. 15, 2365–2369 (2014).
CAS PubMed Google Scholar
Hara, R. & Kino, K. Characterization of novel 2-oxoglutarate dependent dioxygenases converting L-proline to cis-4-hydroxy-L-proline. Biochem. Biophys. Res. Commun. 379, 882–886 (2009).
CAS PubMed Google Scholar
Mori, H., Shibasaki, T., Uozaki, Y., Ochiai, K. & Ozaki, A. Detection of novel proline 3-hydroxylase activities in Streptomyces and Bacillus spp. by regio- and stereospecific hydroxylation of L-proline. Appl. Environ. Microbiol. 62, 1903–1907 (1996).
CAS PubMed PubMed Central Google Scholar
Sheehan, J. C., Mania, D., Nakamura, S., Stock, J. A. & Maeda, K. The structure of telomycin. J. Am. Chem. Soc. 90, 462–470 (1968).
CAS PubMed Google Scholar
Fu, C. et al. Biosynthetic studies of telomycin reveal new lipopeptides with enhanced activity. J. Am. Chem. Soc. 137, 7692–7705 (2015).
CAS PubMed Google Scholar
Koehn, F. E., Longley, R. E. & Reed, J. K. Microcolins A and B, new immunosuppressive peptides from the blue-green alga Lyngbya majuscula. J. Nat. Prod. 55, 613–619 (1992).
CAS PubMed Google Scholar
Schwartz, R. E. et al. Pneumocandins from Zalerion arboricola. I. Discovery and isolation. J. Antibiot. 45, 1853–1866 (1992).
CAS PubMed Google Scholar
Haste, N. M. et al. Activity of the streptogramin antibiotic etamycin against methicillin-resistant Staphylococcus aureus. J. Antibiot. 63, 219–224 (2010).
CAS PubMed PubMed Central Google Scholar
Watanabe, S., Yamada, M., Ohtsu, I. & Makino, K. α-Ketoglutaric semialdehyde dehydrogenase isozymes involved in metabolic pathways of D-glucarate, D-galactarate and hydroxy-L-proline: molecular and metabolic convergent evolution. J. Biol. Chem. 282, 6685–6695 (2007).
CAS PubMed Google Scholar
Watanabe, S. et al. Identification and characterization of D-hydroxyproline dehydrogenase and Δ¹-pyrroline-4-hydroxy-2-carboxylate deaminase involved in novel L-hydroxyproline metabolism of bacteria: metabolic convergent evolution. J. Biol. Chem. 287, 32674–32688 (2012).
CAS PubMed PubMed Central Google Scholar
Visser, W. F., Verhoeven-Duif, N. M. & de Koning, T. J. Identification of a human trans-3-hydroxy-L-proline dehydratase, the first characterized member of a novel family of proline racemase-like enzymes. J. Biol. Chem. 287, 21654–21662 (2012).
CAS PubMed PubMed Central Google Scholar
Watanabe, S. et al. 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. FEBS Open Bio 4, 240–250 (2014).
CAS PubMed PubMed Central Google Scholar
Zhang, X. et al. A unique cis-3-hydroxy-L-proline dehydratase in the enolase superfamily. J. Am. Chem. Soc. 137, 1388–1391 (2015).
CAS PubMed Google Scholar
Gruer, M. J. & Guest, J. R. Two genetically-distinct and differentially-regulated aconitases (AcnA and AcnB) in Escherichia coli. Microbiology 140, 2531–2541 (1994).
CAS PubMed Google Scholar
Williams, C. H. et al. E. coli aconitase B structure reveals a HEAT-like domain with implications for protein-protein recognition. Nat. Struct. Biol. 9, 447–452 (2002).
CAS PubMed Google Scholar
Robbins, A. H. & Stout, C. D. Structure of activated aconitase: formation of the [4Fe-4S] cluster in the crystal. Proc. Natl. Acad. Sci. USA 86, 3639–3643 (1989).
ADS CAS PubMed Google Scholar
Dupuy, J. et al. Crystal structure of human iron regulatory protein 1 as cytosolic aconitase. Structure 14, 129–139 (2006).
CAS PubMed Google Scholar
Grimek, T. L. & Escalante-Semerena, J. C. The acnD genes of Shewenella oneidensis and Vibrio cholerae encode a new Fe/S-dependent 2-methylcitrate dehydratase enzyme that requires prpF function in vivo. J. Bacteriol. 186, 454–62 (2004).
CAS PubMed PubMed Central Google Scholar
Jia, Y., Tomita, T., Yamauchi, K., Nishiyama, M. & Palmer, D. R. Kinetics and product analysis of the reaction catalysed by recombinant homoaconitase from Thermus thermophilus. Biochem. J. 396, 479–485 (2006).
CAS PubMed PubMed Central Google Scholar
Drevland, R. M., Jia, Y., Palmer, D. R. & Graham, D. E. Methanogen homoaconitase catalyzes both hydrolyase reactions in coenzyme B biosynthesis. J. Biol. Chem. 283, 28888–28896 (2008).
CAS PubMed PubMed Central Google Scholar
Fultz, P. N. & Kemper, J. Wild-type isopropylmalate isomerase in Salmonella typhimurium is composed of two different subunits. J. Bacteriol. 148, 210–219 (1981).
CAS PubMed PubMed Central Google Scholar
Yasutake, Y., Yao, M., Sakai, N., Kirita, T. & Tanaka, I. Crystal structure of the Pyrococcus horikoshii isopropylmalate isomerase small subunit provides insight into the dual substrate specificity of the enzyme. J. Mol. Biol. 344, 325–333 (2004).
CAS PubMed Google Scholar
Skala, J., Capieaux, E., Balzi, E., Chen, W. N. & Goffeau, A. Complete sequence of the Saccharomyces cerevisiae LEU1 gene encoding isopropylmalate isomerase. Yeast 7, 281–285 (1991).
CAS PubMed Google Scholar
Makarova, K. S. & Koonin, E. V. Filling a gap in the central metabolism of archaea: prediction of a novel aconitase by comparative-genomic analysis. FEMS Microbiol. Lett. 227, 17–23 (2003).
CAS PubMed Google Scholar
Li, G. & Lu, C. D. Molecular characterization of LhpR in control of hydroxyproline catabolism and transport in Pseudomonas aeruginosa PAO1. Microbiology 162, 1232–1242 (2016).
CAS PubMed Google Scholar
White, C. E., Gavina, J. M., Morton, R., Britz-McKibbin, P. & Finan, T. M. Control of hydroxyproline catabolism in Sinorhizobium meliloti. Mol. MicroBiol. 85, 1133–1147 (2012).
CAS PubMed Google Scholar
Hara, R., Uchiumi, N., Okamoto, N. & Kino, K. Regio- and stereoselective oxygenation of proline derivatives by using microbial 2-oxoglutarate-dependent dioxygenases. Biosci. Biotechnol. Biochem. 78, 1384–1388 (2014).
CAS PubMed Google Scholar
Zhao S., et al. Discovery of new enzymes and metabolic pathways by using structure and genome context. Nature 502, 698–702 (2013).
ADS CAS PubMed PubMed Central Google Scholar
Kumar, R. et al. Prediction and biochemical demonstration of a catabolic pathway for the osmoprotectant proline betaine. MBio 5, e00933–13 (2014).
PubMed PubMed Central Google Scholar
Beinert, H., Kennedy, M. C. & Stout, C. D. Aconitase as iron-sulfur protein, enzyme, and iron-regulatory protein. Chem. Rev. 96, 2335–2374 (1996).
CAS PubMed Google Scholar
Costello, L. C., Liu, Y., Franklin, R. B. & Kennedy, M. C. Zinc inhibition of mitochondrial aconitase and its importance in citrate metabolism of prostate epithelial cells. J. Biol. Chem. 272, 28875–28881 (1997).
CAS PubMed Google Scholar
Frishman, D. & Hentze, M. W. Conservation of aconitase residues revealed by multiple sequence analysis: implications for structure/function relationships. Eur. J. Biochem. 239, 197–200 (1996).
CAS PubMed Google Scholar
Zheng, L., Kennedy, M. C., Beinert, H. & Zalkin, H. Mutational analysis of active site residues in pig heart aconitase. J. Biol. Chem. 267, 7895–7903 (1992).
CAS PubMed Google Scholar
Messerschmidt, A., Huber, R., Poulos, T. & Wieghardt, K. Handbook of Metalloproteins, Vol. I, pp.505–609 Eds. John Wiley & Sons, Ltd., West Sussex, England.
Maltempo, M. M., Moss, T. H. & Cusanovich, M. Magnetic studies on the changes in the iron environment in Chromatium ferricytochrome c’. Biochim. Biophys. Acta 342, 290–305 (1974).
CAS PubMed Google Scholar
Fujii, S. et al. Electron paramagnetic resonance studies of ferric cytochrome c’ from photosynthetic bacteria. Biochim. Biophys. Acta 1251, 161–169 (1995).
PubMed Google Scholar
Meyer, J. Iron-sulfur protein folds, iron-sulfur chemistry, and evolution. J. Biol. Inorg. Chem. 13, 157–170 (2008).
CAS PubMed Google Scholar
Buongiorno, D. & Straganz, G. D. Structure and function of atypically coordinated enzymatic mononuclear non-heme-Fe(II) centers. Coord. Chem. Rev. 257, 541–563 (2013).
CAS PubMed PubMed Central Google Scholar
Lee, E. H., Lee, K. & Hwang, K. Y. Structural characterization and comparison of the large subunits of IPM isomerase and homoaconitase from Methanococcus jannaschii. Acta Crystallogr. D 70, 922–931 (2014).
CAS Google Scholar
Lombo, T. et al. Functional analysis of the small subunit of the putative homoaconitase from Pyrococcus horikoshii in the Thermus lysine biosynthetic pathway. FEMS Microbiol. Lett. 233, 315–324 (2004).
CAS PubMed Google Scholar
Jensen, R. A. Enzyme recruitment in evolution of new function. Annu. Rev. Microbiol. 30, 409–425.
Kobayashi, Y., Ohtsu, I., Fujimura, M. & Fukumori, F. A mutation in dnaK causes stabilization of the heat shock sigma factor σ32, accumulation of heat shock proteins and increase in toluene-resistance in Pseudomonas putida. Environ. MicroBiol. 13, 2007–2017 (2011).
CAS PubMed Google Scholar
Hishinuma, S., Yuki, M., Fujimura, M. & Fukumori, F. OxyR regulated the expression of two major catalases, KatA and KatB, along with peroxiredoxin, AhpC in Pseudomonas putida. Environ. Microbiol. 8, 2115–2124 (2006).
CAS PubMed Google Scholar
Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265–275 (1951).
CAS Google Scholar
Watanabe, S., Tanimoto, Y., Nishiwaki, H. & Watanabe, Y. Identification and characterization of bifunctional proline racemase/hydroxyproline epimerase from archaea: discrimination of substrates and molecular evolution. PLoS One 10, e0120349 (2015).
PubMed PubMed Central Google Scholar
Doi, Y. & Takaya, N. A novel A3 group aconitase tolerates oxidation and nitric oxide. J. Biol. Chem. 290, 1412–1421 (2015).
PubMed Google Scholar
Ito, F., Tamiya., T., Ohtsu, I., Fujimura, M. & Fukumori, F. Genetic and phenotypic characterization of the heat shock response in Pseudomonas putida. MicrobiologyOpen 3, 922–936 (2014).
CAS PubMed PubMed Central Google Scholar
Garvey, G. S., Rocco, C. J., Escalante-Semerena, J. C. & Rayment, I. The three-dimensional crystal structure of the PrpF protein of Shewanella oneidensis complexed with trans-aconitate: insights into its biological function. Protein Sci. 16, 1274–1284 (2007).
CAS PubMed PubMed Central Google Scholar

Download references

Acknowledgements

This work was partially supported by JSPS KAKENHI Grants 25440049 and 16K07297 (to S.W.).

Author information

Authors and Affiliations

Department of Bioscience, Graduate School of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama, 790-8566, Ehime, Japan
Seiya Watanabe & Yasuo Watanabe
Faculty of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama, 790-8566, Ehime, Japan
Seiya Watanabe, Rio Fukuda, Mao Miyazaki & Yasuo Watanabe
Center for Marine Environmental Studies (CMES), Ehime University, 2-5 Bunkyo-cho, Matsuyama, 790-8577, Ehime, Japan
Seiya Watanabe
Department of Bio-molecular Engineering, Graduate School of Science and Technology, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, 606-8585, Kyoto, Japan
Kunihiko Tajima
Faculty of Frontiers of Innovative Research in Science and Technology (FIRST), Konan University, 7-1-20 Minatojima-minamimachi, Chuo-ku, Kobe, 650-0047, Hyogo, Japan
Satoshi Fujii
Faculty of Food and Nutritional Sciences, Toyo University, 1-1-1 Izumino, Itakura-machi, Ora-gun, 374-0193, Gunma, Japan
Fumiyasu Fukumori
Research Institute for Science and Engineering, Waseda University, 3-4-1 Ohkubo, Shinjuku-ku, 169-8555, Tokyo, Japan
Ryotaro Hara & Kuniki Kino
Department of Applied Chemistry, Faculty of Science and Engineering, Waseda University, 3-4-1 Ohkubo, Shinjuku-ku, 169-8555, Tokyo, Japan
Kuniki Kino

Authors

Seiya Watanabe
View author publications
You can also search for this author in PubMed Google Scholar
Kunihiko Tajima
View author publications
You can also search for this author in PubMed Google Scholar
Satoshi Fujii
View author publications
You can also search for this author in PubMed Google Scholar
Fumiyasu Fukumori
View author publications
You can also search for this author in PubMed Google Scholar
Ryotaro Hara
View author publications
You can also search for this author in PubMed Google Scholar
Rio Fukuda
View author publications
You can also search for this author in PubMed Google Scholar
Mao Miyazaki
View author publications
You can also search for this author in PubMed Google Scholar
Kuniki Kino
View author publications
You can also search for this author in PubMed Google Scholar
Yasuo Watanabe
View author publications
You can also search for this author in PubMed Google Scholar

Contributions

S.W. conceived, designed, and performed the experiments, analyzed the data, and wrote the manuscript. K.T. and S.F. carried out EPR measurements, analyzed the data obtained, and wrote some of the manuscript. F.F. contributed to qRT-PCR experiments. R.H. and K.K. synthesized a unique dihydroxyproline compound. R.F. and M.M. prepared the recombinant mutated LhpI proteins. Y.W. contributed to improve the article.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Electronic supplementary material

Supplementary Information

Rights and permissions

This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

Reprints and permissions

About this article

Cite this article

Watanabe, S., Tajima, K., Fujii, S. et al. Functional characterization of aconitase X as a cis-3-hydroxy-L-proline dehydratase. Sci Rep 6, 38720 (2016). https://doi.org/10.1038/srep38720

Download citation

Received: 21 July 2016
Accepted: 11 November 2016
Published: 08 December 2016
DOI: https://doi.org/10.1038/srep38720

This article is cited by

Hydroxyproline in animal metabolism, nutrition, and cell signaling
- Shengdi Hu
- Wenliang He
- Guoyao Wu
Amino Acids (2022)
Crystal structures of aconitase X enzymes from bacteria and archaea provide insights into the molecular evolution of the aconitase superfamily
- Seiya Watanabe
- Yohsuke Murase
- Kunihiko Tajima
Communications Biology (2021)

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Subjects

Abstract

Similar content being viewed by others

Crystal structures of aconitase X enzymes from bacteria and archaea provide insights into the molecular evolution of the aconitase superfamily

An iron (II) dependent oxygenase performs the last missing step of plant lysine catabolism

Structures of fungal and plant acetohydroxyacid synthases

Introduction

Results and Discussion

Identification of substrates and functions

Catalytic properties

Characterization of other AcnXType I enzymes

Putative active sites

Putative iron binding sites

Non-heme iron analysis

Degradation pathway of L-hydroxyproline(s) in P. aeruginosa PAO1

Conclusion

Methods

Plasmid construction for the expression of recombinant proteins

Expression and purification of the recombinant protein

Screening of the proline derivative library for the LhpI protein

Enzyme assay

Identification of reaction products

Non-heme iron analysis

Growth and gene expression analysis of P. aeruginosa PAO1

Sequence comparison

Additional Information

References

Acknowledgements

Author information

Authors and Affiliations

Contributions

Ethics declarations

Competing interests

Electronic supplementary material

Supplementary Information

Rights and permissions

About this article

Cite this article

Share this article

This article is cited by

Hydroxyproline in animal metabolism, nutrition, and cell signaling

Crystal structures of aconitase X enzymes from bacteria and archaea provide insights into the molecular evolution of the aconitase superfamily

Comments

Search

Quick links

Characterization of other AcnX_{Type I} enzymes