trans-3-hydroxy-L-proline dehydratase
Identi?cation and characterization of trans -3-hydroxy-L -proline dehydratase and D 1-pyrroline-2-carboxylate reductase involved in trans -3-hydroxy-L -proline metabolism of
bacteria
Seiya Watanabe a ,?,Yoshiaki Tanimoto a ,Seiji Yamauchi b ,Yuzuru Tozawa b ,Shigeki Sawayama c ,Yasuo Watanabe a
a
Faculty of Agriculture,Ehime University,3-5-7Tarumi,Matsuyama,Ehime 790-8566,Japan b
Proteo-Science Center,Ehime University,3Bunkyo-cho,Matsuyama,Ehime 790-8577,Japan c
Graduate School of Agriculture,Kyoto University,Oiwake-cho,Kitashirakawa,Sakyo-ku,Kyoto 606-8502,Japan
a r t i c l e i n f o Article history:
Received 20January 2014Revised 18February 2014Accepted 19February 2014
Keywords:
Hydroxyproline
trans -3-Hydroxy-L -proline metabolism trans -3-Hydroxy-L -proline dehydratase D 1-Pyrroline-2-carboxylate reductase Convergent evolution of enzyme
a b s t r a c t
trans -4-Hydroxy-L -proline (T4LHyp)and trans -3-hydroxy-L -proline (T3LHyp)occur mainly in colla-gen.A few bacteria can convert T4LHyp to a -ketoglutarate,and we previously revealed a hypothet-ical 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 ?rst 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 D 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 homol-ogous with the mammalian enzyme.On the other hand,Pyr2C reductase encoded to LhpI gene was a novel member of ornithine cyclodeaminase/l -crystallin superfamily,differing from known bacte-rial protein.Furthermore,the LhpI enzymes of A.brasilense and another bacterium showed several different properties,including substrate and coenzyme speci?cities.T3LHyp was converted to pro-line by the puri?ed 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 D 1-piperidine-2-carboxylate in these pathways,and that the T3LHyp pathway is not linked to T4LHyp and L -proline metabolism.
ó2014The Authors.Published by Elsevier B.V.on behalf of the Federation of European Biochemical Societies.This
is an open access article under the CC BY-NC-ND license (https://www.wendangku.net/doc/8517913319.html,/licenses/by-nc-nd/3.0/).
1.Introduction
Hydroxy-L -proline (L -Hyp)has been found in certain proteins,in particular collagen,and in some peptide antibiotics.In mammalian systems,L -proline residue is post-translationally hydroxylated to trans -4-hydroxy-L -proline (T4LHyp)or trans -3-hydroxy-L -proline (T3LHyp)by prolyl 4-hydroxylase (EC 1.14.11.2)and propyl 3-hydroxylase (EC 1.14.11.7),respectively [1].Additionally,it is
known that a few bacterial enzymes directly hydroxylate free L -proline to T4LHyp [2],cis -4-hydroxy-L -proline (C4LHyp)[3]or cis -3-hydroxy-L -proline (C3LHyp)[4].Among several stereoiso-mers of L -Hyp,T4LHyp is the most common in nature.In mammals,T4LHyp is converted to pyruvate and glyoxylate via three interme-diates,D 1-pyrroline-3-hydroxy-5-carboxylate (Pyr3H5C),4-hydroxyglutamate,and 4-hydroxy-3-oxo-glutarate (HOG),by four mitochondrial enzymes as follows:T4LHyp oxidase,Pyr3H5C dehydrogenase (EC 1.5.1.12),aspartate aminotransferase (EC 2.6.1.23),and HOG aldolase (EC 4.1.3.16)[5].On the other hand,over 50years after the discovery of bacteria capable of growing on T4LHyp as a sole carbon source,the enzymes (genes)involved in the hypothetical degradation were recently understood at the molecular level [6,7].In contrast to mammalians,bacteria metabo-lize T4LHyp to a -ketoglutarate through four enzymatic steps (Fig.1A).First,T4LHyp epimerase (EC 5.1.1.8;encoded by LhpA )
https://www.wendangku.net/doc/8517913319.html,/10.1016/j.fob.2014.02.010
2211-5463/ó2014The Authors.Published by Elsevier B.V.on behalf of the Federation of European Biochemical Societies.This is an open access article under the CC BY-NC-ND license (https://www.wendangku.net/doc/8517913319.html,/licenses/by-nc-nd/3.0/).
Abbreviations:L -Hyp,hydroxy-L -proline;T4LHyp,trans -4-hydroxy-L -proline;T3LHyp,trans -3-hydroxy-L -proline;C4LHyp,cis -4-hydroxy-L -proline;C4DHyp,cis -4-hydroxy-D -proline;Pyr4R H2C,D 1-pyrroline-4R -hydroxy-2-carboxylate;C4DHypDH,C4DHyp dehydrogenase;Pyr2C,D 1-pyrroline-2-carboxylate;Pip2C,D 1-piperidine-2-carboxylate;OCD,ornithine cyclodeaminase;LCD,L -lysine cyclodeaminase
?Corresponding author.Tel./fax:+81899469848.E-mail address:irab@agr.ehime-u.ac.jp (S.Watanabe).
e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /f
catalyzes the isomerization of T4LHyp to cis-4-hydroxy-D-proline (C4DHyp),and this is then oxidized to D1-pyrroline-4R-hydroxy-2-carboxylate(Pyr4R H2C)by C4DHyp dehydrogenase(C4DHypDH; EC1.4.99.-).Pyr4R H2C is converted to a-ketoglutaric semialde-hyde(a KGSA)by Pyr4R H2C deaminase(EC3.5.4.22;LhpC)and, in the fourth step,a KGSA is oxidized to a-ketoglutarate by the en-zyme a KGSA dehydrogenase(EC 1.2.1.26;LhpG).Interestingly, there are two types of C4DHypDHs:Pseudomonas aeruginosa and
correspond to B–D.(B)Novel T3LHyp
clusters related to T4LHyp and/or
the box were puri?ed and characterized
S3A).Gray putative genes are sequentially
light-green),T4LHyp(red),C4DHyp(blue
each of puri?ed protein were applied
(For interpretation of the references
S.Watanabe et al./FEBS Open Bio4(2014)240–250241
Azospirillum brasilense,a4b4c4-type enzyme encoded by LhpB (encoding to b-subunit),LhpE(a-subunit)and LhpF genes (c-subunit);Pseudomonas putida;homodimeric-type enzyme en-coded by LhpB gene.This?nding strongly suggests that the T4LHyp pathway clearly evolved convergently in bacteria.LhpABCEFG genes are often clustered together with gene(s)encoding putative amino acid transporter on bacterial genomes(referred to as T4LHyp gene cluster)(Fig.1D).
In the case of T3LHyp,small amounts(one amino acid per 1000amino acids)have been shown to occur in different types of collagen,including collagen I,II and III,but it was found to be particularly abundant in collagen IV(as much as10%of the total L-Hyp content).Although degradation by organism(s)is poorly understood,Visser et al.[8]recently reported that a human C14orf149protein catalyzes the dehydration of T3LHyp to D1-pyrroline-2-carboxylate(Pyr2C)via a putative D2-pyrro-line-2-carboxylate intermediate,although the mechanism of the ability to metabolize T3LHyp by human cells is unclear.Interest-ingly,in spite of different reactions,the T3LHyp dehydratase(EC 4.2.1.77)belongs to the proline racemase superfamily,in which the archetypical proline racemase(EC 5.1.1.4,Ref.[9])and T4LHyp epimerase[10]are also contained(see Fig.S3B).It had been believed that the T3LHyp dehydratase is found only in animals and fungi[8].
Although the metabolic fate of Pyr2C may be conversion to L-proline by NAD(P)H-dependent reductase(Fig.1B),the corre-
sponding gene has not yet been identi?ed.On the other hand, reductase for Pyr2C from bacteria has been already studied.In Pseudomonas strains including P.putida[11]and Pseudomonas syringae[12],D-lysine is metabolized to a-ketoadipate through the so-called‘‘L-pipecolate pathway’’,in which dpkA protein cata-lyzes the second step(conversion of D1-piperidine-2-carboxylate (Pip2C)to L-pipecolate)as a Pip2C reductase(EC 1.5.1.21) (Fig.1B).Indeed,this gene also functions as a Pyr2C reductase (EC1.5.1.1)involved in(hypothetical)D-proline metabolism,in which Pyr2C may be produced from D-proline by(an unknown) D-amino acid oxidase[11].The bifunctional Pyr2C/Pip2C reductase belongs to a novel NAD(P)H-dependent malate/L-lactate dehydro-genase(MDH/LDH)superfamily with no sequence homology to ‘‘orthodox’’MDH/LDH,and shows strict NADPH dependence.It is unclear whether the dpkA(-like)protein functions as Pyr2C reduc-tase in T3LHyp metabolism,because there is no homolog on mam-malian genomes,and P.putida cannot metabolize T3LHyp(see in text).
In this study,we?rst identi?ed that A.brasilense,previously known as a T4LHyp-metabolizing bacterium[6],has the ability to grow on T3LHyp as a sole carbon source,and that the metabolic pathway actually contains T3LHyp dehydratase and Pyr2C reduc-tase,as proposed in mammals.Interestingly,Pyr2C reductase is a novel member of the ornithine cyclodeaminase/l-crystallin super-family,different from known dpkA protein,and there are several signi?cant differences in the enzymatic properties between A.bra-silense and another bacteria:substrate and coenzyme speci?cities. Metabolic networks among T3LHyp,T4LHyp,D-proline and D-ly-sine are also discussed.
2.Results
2.1.Hypothetical metabolic pathway of T3LHyp in A.brasilense
First,we found that among three bacteria capable of metabo-lizing 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?rst 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 struc-tural 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,signi?cant activities of T3LHyp dehydratase and Pyr2C reductase with dual speci?city between NADPH and NADH were found in cell-free extract pre-pared 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 dif-ferent inductivity by carbon sources and coenzyme speci?city.
2.2.Candidates of T3LHyp dehydratase and Pyr2C reductase genes
Although the genome sequence of A.brasilense is unavailable, nucleotide sequences of several genes from this bacterium show very high similarity(>$98%)to those of Burkholderia lata,which was formerly described as Burkholderia sp.183[6].Therefore,a homology search using the Protein-BLAST program was carried out against the genome sequence of https://www.wendangku.net/doc/8517913319.html,ta using C14orf149 (T3LHyp dehydratase)as the probe protein sequence,although it had been believed that only animals and fungi possess this en-zyme,not bacteria[8].Among two homologous proteins(genes) annotated as putative proline racemases,Bcep18194_B1894and Bcep18194_B1660with sequence similarities of29%and44%to C14orf149,respectively,the former corresponded to T4LHyp epi-merase(LhpA)(Fig.1D),whereas the latter possessed two speci?c active sites for T3LHyp dehydratase(see below;Ref.[8])(referred to as LhpH)(Fig.S3A).Therefore,we thought that the LhpH gene was the?rst candidate for a T3LHyp dehydratase.
https://www.wendangku.net/doc/8517913319.html,ta(probably also A.brasilense)possessed one homologous protein(gene)to dpkA from P.putida($40%identity;PP_3591). However,this gene(Bcep18194_B1898;referred to as LhpD)was contained within the T4LHyp gene cluster(Fig.1D),which was up-regulated only by T4LHyp,not T3LHyp,as described above (Fig.2A).On the other hand,further bioinformatics analysis re-vealed that a(putative)LhpH gene from other bacteria such as Col-wellia psychrerythraea34H(CPS_1453)is located within the T4LHyp gene cluster together with one function-unknown protein (gene)annotated as a ornithine cyclodeaminase(OCD;EC4.3.1.12) (referred to as LhpI),instead of LhpD gene(Fig.1D).A gene homol-ogous to CpLhpI gene was also found within the?anking region of AbLhpH gene,and the enzyme reaction by OCD contained Pyr2C as an intermediate(see Fig.5C).Based on these analysis,we selected LhpD and/or LhpI gene as candidates for Pyr2C reductase.
2.3.Preparation of recombinant His6-tag proteins
After cloning all target genes into the vector pETDuet-1,the re-combinant enzymes with attached His6-tags at their N-termini were expressed in Escherichia coli and puri?ed with an Ni2+-chelat-ing af?nity column(PaLhpD was characterized instead of AbLhpD because of its successful expression in E.coli cells,and there is 59.8%identity between the proteins)(Fig.1F).Apparent molecular masses of AbLhpH,CpLhpH,C14orf149,PaLhpD,AbLhpI,and CpLhpI,estimated by SDS–PAGE,were40(37,805.87),40 (40,437.15),44(39,618.72),40(37,164.78),37(33,312.46),and 40(36,181.76)kDa(values in parentheses indicate the calculated molecular mass of the enzyme with His6-tag),and those estimated by analytic gel?ltration were87,98,70,62,78and81kDa,respec-tively(Fig.S2).Therefore,all of these enzymes appear to be dimeric.
242S.Watanabe et al./FEBS Open Bio4(2014)240–250
2.4.Characterization of LhpH protein as bacterial T3LHyp dehydratase
Potential T3LHyp dehydratase activity in LhpH proteins were assayed by the colorimetric method based on the reaction of2-aminobenzaldehyde with Pyr2C[8],described in Section4.Speci?c activities with T3LHyp of AbLhpH,CpLhpH,and C14orf149(as a reference)were19.8,19.3,and7.22U mg proteinà1,respectively. Optimum pH values was also determined by this method:common range of pH8.0–9.5(data not shown).This indicated that the LhpH gene encodes T3LHyp dehydratase(?rst example for bacteria),and that the biochemical properties are similar to the mammalian en-zyme C14orf149.
2.5.Characterization of LhpD as Pyr2C reductase
The puri?ed PaLhpD showed similar reductase activities for both Pyr2C and Pip2C in the presence of NADPH but not NADH: 42.3and32.8U mg proteinà1,respectively(Table1).Although both L-proline and L-pipecolate underwent NADP+-dependent oxi-dization,their k cat/K m values were$500and$350-fold lower than those with Pyr2C and Pip2C,respectively(Table2).Namely,the reaction equilibrium favors the direction toward NADPH-depen-dent reduction.Optimum pH values in reduction and oxidization using Pyr2C and L-proline were pH7.0and pH10.0,respectively (Fig.3A).These substrate and coenzyme speci?cities also corre-sponded to zymogram staining analysis(Fig.3C).Overall,PaLhpD (probably also AbLhpD)showed similar enzymatic properties to dpkA[11,12].Furthermore,it was likely that based on coenzyme speci?city,LhpD corresponds to an enzyme induced by T4LHyp (and C4DHyp)in A.brasilense cells(Fig.2A).
2.6.Characterization of LhpI as novel Pyr2C reductase
First,the LhpI proteins were assayed for OCD activity,with none detected(data not shown).Alternatively,when Pyr2C and NADPH was used as the substrate and coenzyme,respectively,signi?cant
G l L-P D-P T4C4T3D-L
rRNA
carbon sources.Values are the means
transcriptional effect of carbon source on AbLhpI
Table1
Kinetic parameters of AbLhpI,CpLhpI,and PaLhpD in the forward direction.
Enzymes Substrates pH a Coenzymes Speci?c activity(units/mg protein)K m(mM)k cat(minà1)k cat/K m b(minà1mMà1)
AbLhpI Pyr2C 6.5NADPH5840.628±0.04531400±130050100±1400
NADH6000.837±0.08036900±240044100±1400 Pip2C NADPH2200.474±0.0509950±61021100±1010
NADH1770.600±0.0059690±38016200±680 Pyr4S H2C NADPH12.00.491±0.063621±581270±45 CpLhpI Pyr2C NADPH30.2 5.90±0.367470±4701270±2
NADH 4.76 2.79±0.42621±34225±24 Pip2C NADPH0.2917.26±1.2282.9±13.711.4±0.2
NADH0.0464 2.10±0.17 5.27±0.24 2.51±0.09 Pyr4S H2C NADPH0.1748.28±2.4561.3±17.27.43±0.14 PaLhpD Pyr2C7.0NADPH42.30.447±0.0402500±1205600±220 Pip2C38.3 1.57±0.32120±2901350±68
Pyr4S H2C12.10.835±0.109868±711043±48
a pH of potassium phosphate buffer for assay.
b Illustrated in Fig.3D.
S.Watanabe et al./FEBS Open Bio4(2014)240–250243
reduction of activity was observed.Furthermore,L-proline was the active substrate for the NADP+-dependent oxidization reaction,and ratios of Pyr2C to L-proline in k cat/K m were771and356for AbLhpI and CpLhpI,respectively(Tables1and2),suggesting the prefer-ence of the reaction equilibrium to the direction toward NADPH-dependent reduction.Their optimum pH values in reduction and oxidization using Pyr2C and L-proline were pH6.5and pH10.5, respectively(Fig.3A).These properties as a Pyr2C reductase were similar to PaLhpD.
On the other hand,there were also several signi?cant differ-ences in enzymatic properties between AbLhpI and CpLhpI(and PaLhpD).First,the k cat/K m value for Pyr2C of AbLhpI (50,100minà1mMà1)was$350-and8.9-fold higher than those of CpLhpI and PaLhpD,respectively,commonly caused by the high-er k cat values.Second,there was no preference for coenzyme utili-zation between NADPH and NADH in AbLhpI,whereas the k cat/K m value for Pyr2C of CpLhpI in the presence of NADH was5.6-fold lower than that in the presence of NADPH,mainly caused by the 12-fold lower k cat value.Much higher preference for coenzymes was found in the oxidization of L-proline:the ratio of NADPH to NADH in k cat/K m value was161.Third,AbLhpI could utilize Pip2C (and L-pipecolate)in almost the same manner as Pyr2C(L-proline). On the other hand,k cat/K m values for Pip2C and L-pipecolate of CpLhpI(in the presence of NADP+(H))were111-and223-fold low-er than those for Pyr2C and L-proline,mainly caused by90-and51-fold lower k cat values,respectively.To estimate more detailed sub-strate speci?city,several proline analog substrates(because of easy preparation)were used for the NADP+-dependent oxidization reac-tion,in addition of L-proline and L-pipecolate.Only PaLhpD showed 32.8%,14.4%and12.2%activity for T3LHyp,cis-4-hydroxy-L-proline (C4LHyp)and cis-3-hydroxy-L-proline(C3LHyp)as a percent of L-proline,respectively(Fig.3B).Among the corresponding substrates for the NADPH-dependent reduction reaction,we successfully syn-thesized D1-pyrroline-4S-hydroxy-2-carboxylate(Pyr4S H2C)for C4LHyp.The ratio of Pyr2C to Pyr4S H2C of PaLhpD,AbLhpI and CpLhpI(in the presence of NADPH)was5.4,39.4and171,respec-tively,raising the possibility that PaLhpD can utilize Pyr4S H2C as a physiological substrate(see Section3).Such different properties among these three enzymes also corresponded to zymogram stain-ing analysis(Fig.3C).These results suggested that AbLhpI and CpLhpI are bifunctional NAD(P)H-dependent Pyr2C/Pip2C reduc-tase and monofunctional NADPH-preference Pyr2C reductase, respectively,and that Pyr2C reductase activities induced by T3LHyp,D-proline and D-lysine in A.brasilense cells may be derived from LhpI but not LhpD(Fig.2A).
In HPLC analysis(Fig.4),a peak corresponding to T3LHyp was completely eliminated by incubation with puri?ed AbLhpH(or CpLhpH),probably caused by no reaction of Pyr2C with labeling re-agent for amino acid analysis.On the other hand,when T3LHyp was incubated together with puri?ed AbLhpH and AbLhpI(or CpLhpH and CpLhpI)in the presence of NADPH,a novel peak cor-responding to(L-)proline appeared.This suggested that T3LHyp is converted to L-proline by continuous reactions with LhpH and LhpI under physiologically neutral conditions(pH7.0).
2.7.Amino acid sequence analysis of LhpI
As expected from preliminary annotation,LhpI belongs to the OCD/l-crystallin superfamily including the archetype OCD[13], l-crystallin[14],L-alanine dehydrogenase(EC1.4.1.1)[15],L-argi-
nine dehydrogenase[16],L-lysine cyclodeaminase(LCD;EC 4.3.1.28)[17]and tauropine dehydrogenase(EC 1.5.1.23)[18] (Fig.5A).On the other hand,phylogenetic analysis revealed that AbLhpI and CpLhpI have poor relationship not only to any subclass-es of the other members but also each other.Putative amino acid sequences of the LhpI proteins contained essential amino acid res-idues for coenzyme binding(Rossmann-fold motif consisting of Gly-X-Gly-X2-[Ala/Ser],where X indicates any amino acid)and the catalytic triad for binding to a carboxyl group of substrate (Lys-Arg-Asp):Gly137-Thr-Gly-Lys-Gln-Ala142and Lys70-Arg113-Asp294in AbLhpI,respectively(Fig.5B and C).On the other hand, putative amino acid residues,responsible for discrimination be-tween NAD+(H)and NADP+(H),were different not only from other OCD/l-crystallin members but also within LhpI proteins:AbLhpI, Gly162-Thr163;CpLhpI,Gly158-Arg159.
2.8.Gene regulation and disruptant analysis
It is likely that AbLhpH and AbLhpI genes are clustered together with the putative amino acid transporter gene on the genome of A. brasilense(Fig.1D).Northern blot analysis revealed that AbLhpI gene was induced by T3LHyp,D-proline and D-lysine,but not T4LHyp(and C4DHyp)(Fig.2B).To further estimate the physiolog-ical roles of LhpI gene,we carried out gene disruption experiments by introducing a kanamycin-resistant gene(Km r)into AbLhpI gene. The obtained AbLhpIàmutant strain was distinct from the wild-type strain in that T3LHyp,D-proline and D-lysine did not support growth as a sole carbon source.On the other hand,there was no difference in growth on other carbon sources,including T4LHyp, between the two strains.Although we did not analyze AbLhpD in this study,it has been reported that disruption of LhpD gene from Sinorhizobium meliloti had no effect on growth on T4LHyp,in spite of transcriptional induction by T4LHyp(Fig.1D)[19].Overall,these results suggested clearly that LhpI gene is a Pyr2C/Pip2C reductase
Table2
Kinetic parameters of AbLhpI,CpLhpI and PaLhpD in the reverse direction.
Enzymes Substrates pH a Coenzymes Speci?c activity(units/mg protein)K m(mM)k cat(minà1)k cat/K m b(minà1mMà1)
AbLhpI L-Proline10.5NADP+ 5.18 3.63±0.31235±765.0±3.7
NAD+ 5.50 3.99±0.18254±363.6±2.1 L-Pipecolate NADP+ 2.92 6.54±0.96150±1423.0±1.2
NAD+ 2.7214.8±2.1222±2415.0±0.5
CpLhpI L-Proline NADP+0.63018.3±2.465.3±6.9 3.57±0.09
NAD+0.0045118.8±3.80.415±0.0770.0222±0.0004 L-Pipecolate NADP+0.0043580.1±2.8 1.28±0.040.016±0.001
NAD+0.000457N.D.c N.D.N.D.
PaLhpD L-Proline10.0NADP+ 1.9818.5±2.1205±1811.1±0.3 L-Pipecolate0.67934.8±4.0135±14 3.88±0.05
T3LHyp0.490132±26272±53 2.07±0.01
a pH of glycine–NaOH buffer for assay.
b Illustrated in Fig.3E.
c Not determine
d du
e to trace activity.
244S.Watanabe et al./FEBS Open Bio4(2014)240–250
involved in T3LHyp,D-proline and D-lysine metabolism,and that LhpD gene is not related directly to T3LHyp and T4LHyp metabo-lism(see below).
3.Discussion
In this study,we identi?ed T3LHyp pathway consisting of T3LHyp dehydratase and Pyr2C reductase of bacteria.A similar
T3LHyp metabolic pathway may exist in mammals[8],however, this contrasts with T4LHyp metabolic pathways as there are com-plete different in bacteria and mammals[5–7].
On the basis of two speci?c residues at the active sites,proline racemase-like enzymes have been classi?ed into three types:Cys-Cys type(proline racemase and T4LHyp epimerase);Cys-Thr type (T3LHyp dehydratase);Ser-Cys type(function-unknown) (Fig.S3A).Interestingly,a mutant enzyme of T3LHyp dehydratase AbLhpI CpLhpI PaLhpD
111
222
333
S.Watanabe et al./FEBS Open Bio4(2014)240–250245
(C14orf149)with a substitution of threonine to cysteine shows ‘‘T3LHyp epimerase’’activity[8].In the phylogenetic tree (Fig.S3B),T4LHyp epimerase and T3LHyp dehydratase in the same bacteria(LhpA and LhpH,respectively)belong to different subfam-ilies,indicating that dehydratase activity with T3LHyp in this superfamily was acquired once at an early evolutional stage.
In mammalians,Pyr2C is one of the substrates for ketimine reductase(EC1.5.1.25),in which Pip2C and several ketimine com-pounds with neurological functions are also contained[20].It was recently reported that l-crystallin,belonging to the same protein family as LhpI,has ketimine reductase activity[21]:this protein was previously known as an NADPH-dependent thyroid hor-mone-binding protein without enzymatic function[14].However, the puri?ed l-crystallin accounted for only0.19%of total enzyme activity measured in a cell-free extract of the lamb forebrain,and enzyme activity was optimal at acidic pH4.5–5.0,in contrast with neutral pH of LhpI(Fig.3A):it is doubted that this protein physio-logically functions as the major ketimine reductase(and also Pyr2C reductase involved in T3LHyp metabolism)[22].Furthermore,in spite of reaction similarity,bacterial LhpI proteins form a distinct subfamily from l-crystallin in the phylogenetic tree(Fig.5A).Sim-ilar phenomena were also found in OCDs of bacteria and plants [13,23].Based on these insights,we assumed that these subfami-lies appeared by the independent acquisition of substrate speci?c-ity for Pyr2C(and/or Pip2C)rather than divergence from a common ancestor:convergent https://www.wendangku.net/doc/8517913319.html,ly,it is still unclear whether T3LHyp pathways of bacteria and mammalian evolved from the same ancestor(s).
OCD and LCD catalyze unusual cyclization to convert L-orni-thine and L-lysine to L-proline and L-pipecolate via Pyr2C and Pip2C intermediates,with the release of ammonia,respectively(Fig.5C; Refs.[13,17]).Homologous aspartate and glutamate residues,re-lated to cyclization[24],are not found in LhpI(and other OCD/l-crystallin members),con?rming only Pyr2C(and/or Pip2C)reductase activity.In OCD(from P.putida),Asp161forms hydrogen bonds with the20-and30-hydroxyl groups of the NADH ribose moi-ety[13,24](Fig.5B).On the other hand,in l-crystallin(from hu-man),the20-phosphate group of the NADPH ribose moiety interacts with side-chains of Asn168(equivalent to Asp161in OCD),Arg169and Thr170,in which the enzyme favors binding to NADP+(H)with negative charge[14].Such a tendency has been ob-served in other many dinucleotide-binding domains[25].Both Ab-LhpI and CpLhpI possess no homologous Asp residue to Asp161in OCD(substitution to Gly),and the homologous Arg residue to Arg169in l-crystallin is found only in CpLhpI.Site-directed muta-genetic study is in progress to assess the unique coenzyme speci-?city of LhpI at the molecular level.
No homologous gene with known metabolic genes(proteins) involved in the L-pipecolate pathway of D-lysine has been found on the genome of C.psychrerythraea,con?rming that CpLhpI shows no signi?cant Pip2C reductase activity(Tables1and2):this bacte-ria may probably possess no ability to metabolize D-lysine.Such different substrate speci?city from AbLhpI may be related to their distant phylogenetic relationship(only21.8%identity)(Fig.5A): there is a possibility of convergent evolution even within bacterial LhpI proteins,similar to C4DHypDH proteins in T4LHyp metabo-lism[6].
One of the most interesting points in this study is that in A. brasilense,LhpI(but not LhpD),functions as a Pyr2C/Pip2C reduc-tase not only in T3LHyp but also D-proline and D-lysine metabo-lism(Fig.1B),which are good examples of convergent evolution.Indeed,the OCD/l-crystallin superfamily(but not MDH/LDH superfamily including dpkA)contains several enzymes related to proline,lysine and arginine metabolism(Fig.S4).Since the?nal product of T3LHyp metabolism is L-proline,these path-ways might have evolved by duplication and divergence of a common ancestor.Then,what is the physiological role of LhpD? This enzyme can ef?ciently utilize Pyr4S H2C(and C4LHyp)as a substrate(Table1and Fig.3B,D and E).Furthermore,we recently found that C4LHyp and T4DHyp are also substrates of T4LHyp epimerase and C4DHypDH,respectively,by which C4LHyp is con-verted to T4DHyp(via Pyr4S H2C)(Fig.1C)[26].Such L-to D-epi-merization(racemization)of amino acids consisting of two distinct FAD-and NAD(P)+-dependent dehydrogenase(oxidase) is also found in arginine,lysine and proline metabolism from bac-teria(Fig.S4)[16].Therefore,it is likely that LhpD is involved in the degradation of C4LHyp,a compound which is generated by the hydroxylation of free L-proline by bacteria[3].
A.brasilense possesses two separated gene clusters for T4LHyp and T3LHyp metabolism(Fig.1D),which are up-regulated only by each carbon source(Fig.2).These properties may be suitable for the metabolism of T4LHyp and T3LHyp(and C4LHyp)produced by direct hydroxylation of free L-proline,as described in Section1: in fact,the hydroxylase is often found in soil bacteria that?x nitro-gen(so-called rhizobia;Ref.[3]),similar to A.brasilense.In contrast, the homologous gene clusters of C.psychrerythraea,a marine bac-teria,are combined(Fig.1D):this gene cluster may be induced by both T4LHyp and T3LHyp.If this hypothesis is true,it is likely that gene regulation is advantageous for the utilization of much marine collagen,because of the co-production of T4LHyp and T3LHyp.
Since post-translational hydroxylation of L-proline residues is almost speci?c to collagen protein,L-Hyp(s)provides an impor-tant marker to directly measure collagen content in several sam-ple types,including foods and tissue?brosis.Furthermore,L-Hyp(s)in urine and serum has been focused on as a signi?cant biomarker for bone resorption and many human diseases(urinal T3LHyp for cancer;Ref.[27]).On the other hand,the most popu-lar HPLC method for determination of L-Hyp(s)is time-consuming and requires expensive and large apparatus and organic
solvent, 246S.Watanabe et al./FEBS Open Bio4(2014)240–250
S.Watanabe et al./FEBS Open Bio4(2014)240–250247
and L-Hyp(s)(and L-proline),a cyclic imino acid,cannot react with a general labeling reagent for amino acids.Alternatively, an enzymatic method for the T3LHyp dehydratase assay,de-scribed in Section4,would be helpful for conventional detection of T3LHyp in in vivo samples.This line of study is in progress in our laboratory[28].
4.Experimental procedures
4.1.Materials
T3LHyp was purchased from Kanto Chemical(Tokyo,Japan). C4LHyp,L-pipecolate,and D-pipecolate were obtained from Tokyo Chemical Industry(Tokyo,Japan).T4DHyp and C3LHyp were from Sigma Aldrich(USA).T4LHyp,C4DHyp,L-proline,and D-proline were from Wako Pure Chemical Industries(Osaka,Japan).
4.2.General procedures
Basic recombinant DNA techniques were performed as de-scribed by Sambrook et al.[29].Bacterial genomic DNA was pre-pared using a DNeasy Tissue Kit(Qiagen).PCR was carried out using a GeneAmp PCR System2700(Applied Biosystems)for30cy-cles in50l L reaction mixture containing1U of KOD FX DNA poly-merase(TOYOBO),appropriate primers(15pmol)and template DNA under the following conditions:denaturation at98°C for 10s,annealing at50°C for30s and extension at68°C for time periods calculated at an extension rate of1kbp minà1.DNA sequencing was carried out using the BigDye Cycle Sequencing Kit ver.3.1(Applied Biosystems)and appropriate primers with the Genetic Analyzer3130(Applied Biosystems).Protein concen-trations were determined by the method of Lowry et al.[30]with bovine serum albumin as the standard.SDS–PAGE was performed as described by Laemmli[31].Amino acids were identi?ed using an amino acid analyzer(L-8900;Hitachi,Tokyo,Japan)using commer-cial standards(Wako).
4.3.Substrates
Pyr2C was enzymatically synthesized from T3LHyp with C14orf149.The reaction mixture(10mL)consisted of50mM potassium phosphate buffer(pH7.0)and10mM T3LHyp.After the addition of$20mg of puri?ed C14orf149,the mixture was left at30°C overnight.For enzymatic synthesis of Pip2C and Pyr4S H2C,
a reaction mixture(10mL)consisting of50mM Tris-HCl buffer(pH
9.0),10mM D-pipecolate(for Pip2C)or T4DHyp(for Pyr4S H2C), 0.02mM phenazine methosulfate(PMS),and$20mg puri?ed Ab-LhpBEF(C4DHypDH)was incubated with shaking at30°C over-night in the dark.Pyr2C,Pip2C,and Pyr4S H2C in each reaction mixture were puri?ed using a Dowex1?2Clàform(100–200mesh)resin column,described previously[11].
4.4.Bacterial strain,culture conditions and preparation of cell-free extracts
A.brasilense ATCC29145and P.aeruginosa PAO1were cultured aerobically with vigorous shaking at30°C in minimal medium [7]supplemented with30mM carbon source.C.psychrerythraea 34H was grown at8°C in Marine Broth(Difco2216).The grown cells were harvested by centrifugation at30,000g for20min,sus-pended in50mM Tris-HCl(pH8.0),and disrupted by sonication for20min at appropriate intervals on ice using Ultra Sonic Disrup-tor Model UR-200P(TOMY SEIKO Co.,Ltd.,Tokyo,Japan)and then centrifuged at108,000g for20min at4°C to obtain cell-free extracts.4.5.Plasmid construction for expression of recombinant proteins
Primer sequences used in this study are shown in Table S1.In this report,the pre?xes Ab(A.brasilense),Pa(P.aeruginosa),Cp (C.psychrerythraea)and Pp(P.putida)have been added to gene symbols or protein designations when required for clarity.PaLhpD (PA1252),CpLhpH(CPS_1453),and CpLhpI genes(CPS_1455)were ampli?ed by PCR using primers containing appropriate restriction enzyme sites at the50-and30-ends and genome DNA of P.aerugin-osa or C.psychrerythraea as a template.C14orf149gene was ob-tained from Human cDNA clone AK058165(NITE Biological Resource Center(NBRC),Chiba,Japan).AbLhpH and AbLhpI genes were ampli?ed by PCR using primers designed from putative pro-line racemase(Bcep18194_B1660)and OCD genes (Bcep18194_B1663)from https://www.wendangku.net/doc/8517913319.html,ta and genome DNA of A.brasilense as a template,and the ampli?ed products were sequenced.The nucleotide sequences of AbLhpH and AbLhpI genes were submitted to GenBank with accession numbers GenBank:AB894494and GenBank:AB894495,respectively.
Each ampli?ed DNA fragment was introduced into BamHI-Hin-dIII sites(for PaLhpD,AbLhpH,AbLhpI and CpLhpI genes)or BamHI-PstI(for C14orf149and CpLhpH genes)in pETDuet-1(Novagen),a plasmid vector for conferring N-terminal His6-tag on expressed proteins,to obtain pET/PaLhpD,pET/AbLhpH,pET/AbLhpI,pET/ CpLhpH,pET/CpLhpI,and pET/C14orf149.The?ve former and pET/C14orf149were transformed into E.coli strains BL21(DE3) and BL21(DE3)-RIL(Novagen),respectively.
4.6.Expression and puri?cation of His6-tagged recombinant proteins
E.coli harboring the expression plasmid for His6-tagged pro-teins was grown at37°C to a turbidity of0.6at600nm in Super broth medium(pH7.0,12g tryptone,24g yeast extract,5mL glyc-erol,3.81g KH2PO4,and12.5g K2HPO4per liter)containing50mg/ liter ampicillin.After the addition of1mM isopropyl-b-D-thioga-lactopyranoside(IPTG),the culture was further grown for6h at 37°C(for C14orf149and AbLhpH)or for18h at18°C(for PaLhpD, AbLhpI,CpLhpH and CpLhpI)to induce the expression of His6-tagged protein.Cells were harvested and resuspended in Buffer A (50mM sodium phosphate buffer(pH8.0)containing300mM NaCl and10mM imidazole).The cells were then disrupted by son-ication,and the solution was centrifuged.The supernatant was loaded onto a Ni-NTA Super?ow column(Qiagen)equilibrated with Buffer A linked to the BioAssist eZ system(TOSOH).The col-umn was washed with Buffer B(50mM sodium phosphate buffer (pH8.0)containing300mM NaCl,10%(v/v)glycerol,and50mM imidazole).The enzymes were then eluted with Buffer C(pH8.0, Buffer B containing250mM imidazole instead of50mM imidaz-ole),concentrated by ultra?ltration with Centriplus YM-30(Milli-pore),dialyzed against50mM Tris–HCl buffer(pH8.0) containing50%(v/v)glycerol,and stored atà35°C until use.
The native molecular mass of recombinant proteins was esti-mated by gel?ltration,which was carried out using HPLC with a Multi-Station LC-8020model II system(TOSOH)at a?ow rate of 1mL minà1.The puri?ed enzyme($10mg mLà1)was loaded onto a TSKgel G3000SWXL column(TOSOH)equilibrated with50mM Tris–HCl buffer(pH8.0).A high molecular weight gel?ltration cal-ibration kit(GE Healthcare)was used as a molecular marker.
4.7.Enzyme assay
All enzyme assays were performed at30°C.
T3LHyp dehydratase was assayed spectrophotometrically in the coupling system with PaLhpD(NADPH-dependent Pyr2C reduc-tase)using a Shimadzu UV-1800spectrophotometer(Shimadzu GLC Ltd.,Tokyo,Japan).The reaction mixture consisted of50mM
248S.Watanabe et al./FEBS Open Bio4(2014)240–250
Tris–HCl(pH8.0),0.15mM NADPH and10l g puri?ed PaLhpD.The reaction was started by the addition of100mM T3LHyp(100l L) with a?nal reaction volume of1mL.One unit of enzyme activity refers to1l mol NADPH produced/min.K m and k cat values were calculated by a Lineweaver–Burk plot.The enzyme was alterna-tively assayed by the colorimetric method based on the reaction of2-aminobenzaldehyde with Pyr2C,which yields a yellow reac-tion product[8].This method was used for the determination of optimum pH for the activity.
Pyr2C/Pip2C reductase was assayed routinely in the direction of Pyr2C reduction by measuring the oxidization of NAD(P)H at 340nm.The standard assay mixture contained10mM Pyr2C(or Pip2C)in50mM potassium phosphate(pH6.5for AbLhpI and CpLhpI,or pH7.0for PaLhpD)buffer.The reactions were started by the addition of100l L of a1.5mM NAD(P)H solution to a?nal volume of1mL.To assay the reverse reaction,the reaction mixture consisted of50mM Glycine–NaOH(pH10.0for PaLhpD or pH10.5 for AbLhpI and CpLhpI)and10mM L-proline(or L-pipecolate).The reaction was started by the addition of15mM NAD(P)+(100l L) with a?nal reaction volume of1mL.One unit of enzyme activity refers to1l mol NAD(P)H produced/min.Potential OCD activity in LhpI was assayed by the method described previously[23].
T4LHyp epimerase,C4DHypDH,Pyr4R H2C deaminase,and a KGSA dehydrogenase,involved in T4LHyp metabolism,were as-sayed by the method described previously[7].If necessary,recom-binant AbLhpBEF[26]was used as a coupling enzyme of the epimerase assay as C4DHypDH,instead of recombinant PaLhpBEF.
4.8.Reaction product analysis
Puri?ed AbLhpH and AbLhpI or CpLhpH and CpLhpI(each 10l g)were added to50mM Tris–HCl buffer(pH8.0)containing 10mM T3LHyp and0.15mM NADPH(1mL).After incubation at 30°C overnight,each enzyme product was then analyzed by Hit-achi L-8900amino acid analyzer(Tokyo,Japan),using ion ex-change chromatography followed by post-column derivatization with ninhydrin.Retention times of T3LHyp and L-proline(potential product)were appropriately8.6and33min,respectively.
4.9.Zymogram staining analysis
Puri?ed PaLhpD,AbLhpI and CpLhpI were separated at4°C on non-denaturing PAGE with10%(w/v)gel,which was performed by omitting SDS and2-mercaptoethanol from the solution used in SDS–PAGE.The gels were then soaked in10mL staining solution consisting of50mM Glycine–NaOH(pH10),0.25mM nitroblue tetrazolium(NBT),0.06mM PMS,10mM substrate(L-proline or L-pipecolate),and15mM NAD(P)+at room temperature for
15min in the dark.Dehydrogenase activity appeared as a dark blue band.
4.10.Northern blot analysis
A.brasilense cells were cultured at30°C to the mid-log phase (OD600=0.6–0.8)in minimal medium containing30mM carbon source,and harvested by centrifugation.Total RNA preparation was isolated using the SV Total RNA Isolation Kit(Promega,Madi-son,WI,USA)according to the manufacturer’s instructions.The isolated RNA(4l g)was subjected to electrophoresis on1.2%(w/ v)agarose gel containing0.66M formaldehyde,and blotted to Hy-bond-N(GE Healthcare)by capillary transfer using10?SSC as a transfer buffer(1?SSC is15mM sodium citrate(pH7.0),and 0.15M NaCl).The blotted?lter was cross-linked in a UV cross-lin-ker CX-2000(Ultra-Violet Products,Ltd.).A double-stranded probe DNA was labeled with digoxigenin-11-dUTP and hybridized using a DIG-High Prime DNA labeling and detection starter kit(Roche Applied Science).Membrane was visualized using a nitro blue tet-razolium/5-bromo-4-chloro-3-indolyl phosphate reagent detec-tion system(Roche Applied Science).
4.11.Target disruption of AbLhpI gene
The Tn5-derived SacII1.3-kbp kanamycin resistance(Km r)cas-sette was ampli?ed by PCR using pUC4K(GE Healthcare)as a tem-plate and two primers P13and P14(Table S1),and inserted into the single SacII site in the coding sequence of AbLhpI gene of pET/Ab-LhpI to yield pLhpI::Km.To introduce the restriction site for MfeI at the50-and30-end of the DNA fragment containing the Km r gene in the AbLhpI gene,PCR was carried out using pLhpI::Km as a tem-plate and two primers P15and P16.The2.2-kbp MfeI DNA frag-ment was subcloned into EcoRI site in a chloramphenicol resistance(Cm r)cassette of the suicide vector pSUP202[32]to yield pSUP/LhpI::Km. E.coli S17-1[32]was transformed with pSUP/LhpI::Km,and then the transformant was further mobilized to A.brasilense by biparental mating.The transconjugants were se-lected on a minimal medium agar plate supplemented with5g so-dium malate and25l g kanamycin per liter using Km r(the presence of Km r cassette)and Tc S(loss of pSUP202)phenotypes. The construction was con?rmed by genomic PCR.
4.12.Amino acid sequence alignment and phylogenetic analysis
Protein sequences were analyzed using the Protein-BLAST and Clustal W program distributed by DDBJ(DNA Data Bank of Japan) (www.ddbj.nig.ac.jp).The phylogenetic tree was produced using the TreeView1.6.1.program.
5.Database
Nucleotide sequence data are available in the DDBJ/EMBL/ GenBank databases under the accession number(s)GenBank: AB894494and GenBank:AB894495for T3LHyp dehydratase and Pyr2C reductase genes from A.brasilense.
Acknowledgements
This work was partially supported by a Grant-in-Aid for Scien-ti?c Research from the Ministry of Education,Culture,Sports,Sci-ence and Technology in Japan(25440049)(to S.W.),the A-STEP feasibility study program(AS242Z00554M)from the Japan Science and Technology Agency(JST)(to S.W.),and Hokuto Foundation for Bioscience(to S.W.).We thank Prof.Miyuki Kawano-Kawada(Inte-grated Center for Sciences(INCS),Ehime University)for amino acid analysis.Our thanks are extended especially to Profs.Hiroshi Tak-agi and Iwao Ohtsu,and Dr.Yusuke Kawano(Nara Institute of Technology)for invaluable advice.
Appendix A.Supplementary data
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