In a previous article, a gene cluster related to this path-way was identified, consisting of the genes encoding the four metabolic enzymes l-rhamnose-1-dehydrogenase LRA1, l-rhamnono-c-la
Trang 1metabolism – an alternative L-rhamnose pathway of
Sphingomonas sp.
Seiya Watanabe1,2,3and Keisuke Makino1,2,3,4
1 Institute of Advanced Energy, Kyoto University, Japan
2 New Energy and Industrial Technology Development Organization, Gokasho, Uji, Kyoto, Japan
3 CREST, Japan Science and Technology Agency, Gokasho, Uji, Kyoto, Japan
4 Innovative Collaboration Center, Kyoto University, Japan
Microorganisms can utilize pentoses and deoxyhexoses
as their sole carbon source There are generally two
pathways for the metabolism of these sugars, one with
phosphorylated intermediates and the other without such intermediates The former pathways of bacteria and⁄ or fungi have been studied extensively Many
Keywords
Entner–Doudoroff pathway; gene cluster;
L -rhamnose; metabolic evolution;
Sphingomonas sp.
Correspondence
S Watanabe, Institute of Advanced Energy,
Kyoto University, Gokasho, Uji, Kyoto
611-0011, Japan
Fax: +81 774 38 3524
Tel: +81 774 38 3596
E-mail: irab@iae.kyoto-u.ac.jp
(Received 30 October 2008, revised 9
December 2008, accepted 5 January 2009)
doi:10.1111/j.1742-4658.2009.06885.x
Several bacteria, including Azotobacter vinelandii, possess an alternative pathway of l-rhamnose metabolism, which is different from the known bacterial pathway In a previous article, a gene cluster related to this path-way was identified, consisting of the genes encoding the four metabolic enzymes l-rhamnose-1-dehydrogenase (LRA1), l-rhamnono-c-lactonase (LRA2), l-rhamnonate dehydratase (LRA3) and l-2-keto-3-deoxyrhamno-nate (l-KDR) aldolase (LRA4), by which l-rhamnose is converted into pyruvate and l-lactaldehyde, through analogous reaction steps to the well-known Entner-Doudoroff (ED) pathway In this study, bioinformatic analysis revealed that Sphingomonas sp possesses a gene cluster consisting
of LRA1–3 and two genes of unknown function, LRA5 and LRA6 LRA5 catalyzed the NAD+-dependent dehydrogenation of several l-2-keto-3-de-oxyacid-sugars, including l-KDR Furthermore, the reaction product was converted to pyruvate and l-lactate by LRA6; this is different from the pathway of Azotobacter vinelandii Therefore, LRA5 and LRA6 were assigned as the novel enzymes l-KDR 4-dehydrogenase and l-2,4-diketo-3-deoxyrhamnonate hydrolase, respectively Interestingly, both enzymes were phylogenetically similar to l-rhamnose-1-dehydrogenase and d-2-keto-3-deoxyarabinonate dehydratase, respectively, and the latter was involved in the archeal nonphosphorylative d-arabinose pathway, which is partially analogous to the ED pathway The introduction of LRA1–4 or LRA1–3, LRA5 and LAR6 compensated for the l-rhamnose-defective phenotype of
an Escherichia coli mutant Metabolic evolution and promiscuity between the alternative l-rhamnose pathway and other sugar pathways analogous
to the ED pathway are discussed
Abbreviations
COG, cluster of orthologous groups of proteins; D -KDA, D -2-keto-3-deoxyarabinonate; D -KGD, D -2-keto-3-deoxygluconate; ED, Entner– Doudoroff; FAH, fumarylacetoacetate hydrolase; L -DKDR, L -2,4-diketo-3-deoxyrhamnonate; L -KDF, L -2-keto-3-deoxyfuconate; L -KDL, L -2-keto-3-deoxylyxonate; L -KDM, L -2-keto-3-deoxymannonate; L -KDR, L -2-keto-3-deoxyrhamnonate; LRA1, L -rhamnose-1-dehydrogenase; LRA2,
L -rhamnono-c-lactonase; LRA3, L -rhamnonate dehydratase; LRA4, L -2-keto-3-deoxyrhamnonate aldolase; LRA5, L -2-keto-3-deoxyrhamnonate-(4)-dehydrogenase; LRA6, L -2,4-diketo-3-deoxyrhamnonate hydrolase; MhpD, 2-oxopent-4-enoate hydratase; npED, nonphosphorylative Entner–Doudoroff; SDR, short-chain dehydrogenase ⁄ reductase; aKGSA, a-ketoglutaric semialdehyde.
Trang 2bacteria, including Escherichia coli, also metabolize
l-rhamnose (l-6-deoxymannose) through this type of
pathway, using enzymes consisting of l-rhamnose
isomerase (EC 5.3.1.14), rhamnulokinase (EC 2.7.1.5),
and rhamnulose-1-phosphate aldolase (EC 4.1.2.19)
[1] The l-lactaldehyde obtained, together with
dihydroxyacetone phosphate, is further converted to
l-lactate or 1,2-propanediol by l-lactaldehyde
dehydro-genase (EC 1.2.1.22) [2] and lactaldehyde : propanediol
oxidoreductase [EC 1.1.1.77(55)] [3] under aerobic and
anaerobic conditions, respectively
The pathways without phosphorylated intermediates
are classified into two groups, in which the sugar is
commonly converted into a
dl-2-keto-3-deoxyacid-sugar through the participation of dehydrogenase,
lactonase and dehydratase enzymes (schematic
reac-tions a–c in Fig 1) (each enzyme is referred to as ‘E’ below) In the ‘type I pathway’ of d-glucose [4,5],
d-galactose [6], d-fucose [7–10] and l-arabinose [11], the dl-2-keto-3-deoxyacid-sugar is cleaved through an aldolase reaction (schematic reaction e) to the appro-priate aldehyde and pyruvate as well as to the Entner– Doudoroff (ED) pathway Although most metabolic genes have not yet been identified, except for the ‘non-phosphorylative ED (npED) pathway’ in Archaea, a previous article [12] recently characterized this type of
l-rhamnose pathway in the fungi Pichia stipitis and Debaryomyces hansenii (E12–E15) and the bacterium Azotobacter vinelandii (E16–E19) In this pathway,
l-rhamnose is converted into pyruvate and l-lactalde-hyde via l-rhamnono-c-lactone and l-rhamnonate, by the consecutive action of the enzymes
l-rhamnose-D-Galactose L-Fucose D-Fucose L-Arabinose Pathway Hexaric acids
L-Arabinose
D-Arabinose II
D-Xylose
71
COG2706 COG3386 COG3618 COG2220
COG0129 COG4948
COG0800 COG0329 COG3836 COG0179
COG1012 COG0364
COG1063 COG4993 COG0673 COG1028 COG0667
Glyceraldehyde 3P D-Glyceraldehyde L-Lactate D-Lactaldehyde Glycolaldehyde Final product
α-Ketoglutarate
g 52 55 60 65 70 74 79 84
Hexaric acids L-Rhamnose I
Tartronate semialdehyde
L-Lactaldehyde L-Lactate
Schematic reactions
ED npED
Gluconate
5
Final product (pyruvate +) Glyceraldehyde 3P D-Glyceraldehyde – – –
20
D -Galacturonate
B E B B B Domain B B B B B A B A
B
E B
B A B B B
B
E
b – –
31 34 38 43 47
57 62 67
76 81
–
13 17
b 2
10
21
–
c
32 35 39 44 48
50 53 58 63 68 72 77 82
25
14 18 22
c 3 6
28
f
36 41 45 49
51 54 59 64 69 73 78 83
26
15 19 24
e 4 7
29
–
40 – –
– – – – – – – –
– –
– – 23
d – –
a – –
30 33 37 42 46
56 61 66
75 80
–
12 16
a 1
8 9 11
27 III
III I
I
PPP PPP PPP
ED
Fig 1 Comparison of sugar metabolic
path-ways analogous to the ED pathway in
bacte-ria (B), eukaryotes (E), and Archaea (A).
Sugar is commonly metabolized through
the participation of sugar dehydrogenase (a),
lactone-sugar hydrolase (lactonase) (b),
acid-sugar dehydratase (c),
2-keto-3-deoxyacid-sugar dehydrogenase (d), aldolase (e), and
dehydratase (f) for 2-keto-3-deoxyacid-sugar,
and aKGSA dehydrogenase (g) Colored
COGs are homologous to each other, and
white indicates that the metabolic gene has
not yet been identified Numbers (1–84)
correspond to enzymes catalyzing each
reaction (listed in Table S1), which are
referred to as ‘E’ in the text In this study,
an alternative L -rhamnose pathway including
E20–E24 was focused on (indicated by
cir-cles) The reactions of E24 and E41 can be
assigned as equivalent to e and f (see text).
Trang 31-dehydrogenase (LRA1; EC 1.1.1.173),
l-rhamnono-c-lactonase (LRA2; EC 3.1.1.65), l-rhamnonate
dehydratase (LRA3; EC 4.2.1.90), and
l-2-keto-3-deoxyrhamnonate (l-KDR) aldolase (LRA4) (referred
to as the ‘aldolase pathway’ in this article) (Fig 2A)
Furthermore, the metabolic fate of l-lactaldehyde is
dehydrogenation by l-lactaldehyde dehydrogenase
(EC 1.2.1.22), which is similar to the known bacterial
pathway as described above [13] Interestingly, there
is no evolutionary relationship between the l-KDR
aldolases from fungi and bacteria (E15 and E19)
l-Lactaldehyde dehydrogenases from fungi and bacte-ria belong to different subfamilies in the aldehyde dehydrogenase superfamily, and show distinct coen-zyme and substrate specificities These findings indicate that the pathways have evolved independently in spite
of the homologous schematic conversion of l-rham-nose The ‘type II pathway’ without phosphorylated intermediates corresponds to an alternative pathway of
d-arabinose [14], l-arabinose [15–17], and d-xylose [18] In these pathways, the dl-2-keto-3-deoxypento-nate intermediate is converted to a-ketoglutarate via
L -Rhamnose:H + symporter EAM07803 EAM07804 EAM07805 EAM07806 EAM07807 EAM07808 EAM07809 EAM07810
Sugar transporter Sugar channel
Azotobacter vinelandii
EAT09360 EAT09361 EAT09362 EAT09364
L -Rhamnose:H + symporter EAT09366 β-Xylosidase
EAT09367 EAT09363
EAT09365
Sphingomonas sp SKA58
(NBRC101715)
Pichia stipitis ABN68404 ABN68405 ABN68602 ABN68603
LRA1 LRA3 LRA6 LRA5 LRA2
LRA4 LRA1 LRA2
LRA3
Debaryomyces hansenii CAG87577 CAG87576 CAG87575 CAG87574
LADH
Chr 2 ABN64318
LADH CAG90160 Chr G
Escherichia coli
yfaU yfaV yfaW Transporter
Chr8
Chr E
L -Rhamnose
L -Rhamnono- γ-lactone
L -Rhamnonate
L -2-Keto-3-deoxyrhamnonate ( L -KDR)
L -Rhamnose 1-dehydrogenase
( LRA1, EC 1.1.1.173)
L -Rhamnono-γ-lactonase
( LRA2, EC 3.1.1.65)
L -Rhamnonate dehydratase
( LRA3, EC 4.2.1.90)
NAD(P) +
NAD(P)H
H 2 O
H 2 O
Pyruvate L -Lactaldehyde
L -KDR aldolase
( LRA4)
NAD + NADH
L -2,4-Diketo-3-deoxyrhamnonate
( L -DKDR)
Pyruvate L -Lactate
L -DKDR hydrolase ( LRA6)
L -KDR 4-dehydrogenase ( LRA5)
Aldolase pathway Diketo-hydrolase pathway
L -Lactate
L -Lactaldehyde dehydrogenase ( LADH, EC 1.2.1.22)
NAD +
NADH
A
B
AAM43286 AAM43287 AAM43288 AAM43289 AAM43290 AAM43291 AAM43292
Sugar transporter
L -Fucopyranoside mutarotase
C
Fig 2 The alternative L -rhamnose pathway (A) and schematic gene clusters (B) P stipitis, D hansenii and A vinelandii possess the ‘aldol-ase pathway’, in which L -rhamnose is converted into pyruvate and L -lactaldehyde L -Lactaldehyde dehydrogenase then produces L -lactate from L -lactaldehyde in this pathway In Sphingomonas sp., the L -KDR intermediate is alternatively converted into pyruvate and L -lactate via
L -DKDR (diketo-hydrolase pathway) Chr n indicates chromosome number Homologous genes are indicated in the same color, and corre-spond to Fig 1 Gray putative genes are similar in sequence to other L -rhamnose-related enzymes involved in sugar uptake (C) Schematic gene cluster related to the alternative L -fucose pathway of bacteria [33].
Trang 4a-ketoglutaric semialdehyde (aKGSA) by
dl-2-keto-3-deoxypentonate dehydratase (EC 4.1.2.18; E59, E64,
E69, E73, E78, and E83) and aKGSA dehydrogenase
(EC 1.2.1.26; E60, E65, E70, E74, E79, and E80)
(sche-matic reactions e and f, respectively, in Fig 1)
aKGSA is also produced from hexaric acids
(d-gluca-rate and d-galacta(d-gluca-rate) via d-2-keto-3-deoxygluca(d-gluca-rate
by two successive dehydration reactions in bacteria
(E50–E52 and E53–E55) [19]
Most of these alternative pathways of sugar
metabo-lism have been described on the basis only of enzyme
activities in cell-free extracts from experiments
performed several decades ago It could be useful to
identify a set of the metabolic genes (enzymes) for the
enzymatic synthesis of unavailable specific
intermedi-ates, in particular dl-2-keto-3-deoxyacid-sugars In this
regard, transcriptomic and⁄ or proteomic analysis have
significant advantages, as shown in the alternative
d-arabinose pathway of Archaea [14] We, alternatively,
focused on the following two insights: (a) the equivalent
reaction step-catalyzing metabolic enzymes involved in
sugar pathways analogous to the ED pathway are
classi-fied into limited numbers of the known protein families,
cluster of orthologous groups of proteins (COG)
(Fig 1); (b) metabolic genes often form a single gene
cluster in the genomes of bacteria and Archaea
(Fig 2B) Accordingly, homology searches were carried
out using the known metabolic genes (Table S1) against
the genomes of microorganisms, and
‘semi-automati-cally’ selected a set of four potential metabolic genes,
LRA1–4, involved in the alternative l-rhamnose
path-way described above [12], suggesting that this approach
may be helpful in identifying unknown sugar pathways
analogous to the ED pathway, even if the phenotype of
the microorganism is not available
This study further developed this possibility and
revealed that in Sphingomonas sp., a
phenotype-unknown but genome sequence-available bacterium,
l-rhamnose is converted into pyruvate and l-lactate
(but not l-lactaldehyde) via four nonphosphorylated
intermediates by five metabolic enzymes (genes), which
differed partially from the aldolase pathway
Compari-sons between the novel l-rhamnose pathway and other
sugar metabolic pathways and the substrate promiscuity
in the metabolic enzymes are also described
Results
Gene cluster related toL-rhamnose metabolism
in Sphingomonas sp
Several significant insights were obtained about sugar
pathways analogous to the ED pathway, including the
alternative l-rhamnose pathways of fungi and bacteria (Fig 1) Therefore, an extended bioinformatic analysis was carried out, and an interesting gene cluster related
to putative sugar metabolism was found in the genome
of Sphingomonas sp SKA58 (Fig 2B) In this article, the prefixes Ps (P stipitis), Dh (D hansenii), Av (A vinelandii) and Sp (Sphingomonas sp.) have been added to gene symbols or protein designations when required for clarity
When compared with the LRA1–4 gene clusters
of fungi and bacteria, the gene EAT09362 of Sphingo-monas sp was homologous to the LRA3 gene encod-ing l-rhamnonate dehydratase (COG4948, enolase superfamily); there was 80.2% identity with AvLRA3 Furthermore, gene EAT09365 belonged to the same COG group as the LRA2 gene encoding l-rhamnono-c-lactonase (COG3618, a⁄ b hydrolase fold enzymes)
On the other hand, the gene cluster of Sphingomonas
sp SKA58 contained the genes encoding two putative short-chain dehydrogenase⁄ reductase (SDR) family
EAT09364 (SpLRA5) (Fig 2B) As the former showed higher amino acid sequence homology to other LRA1 proteins than the latter (66.7% and 25.3% identity with AvLRA1, respectively), the enzyme function of EAT09360 is likely to play a role
as an l-rhamnose-1-dehydrogenase (see below) On the other hand, there was no homolog to the EAT09363 gene in the LRA1–4 gene cluster: COG0179, 2-oxopent-4-enoate hydratase (MhpD) family These results indicated that the gene cluster of Sphingomonas sp SKA58 may be responsible for non-phosphorylative sugar metabolism, in which the sche-matic conversion of the l-2-keto-3-deoxyacid-sugar intermediate is different from the aldol cleavage by
l-KDR aldolase In this study, we used Sphingomonas
sp NBRC 101715 instead of Sphingomonas sp SKA58 as a target microorganism; between these, there is 99.8% identity in 16S rRNA sequence We were successful in amplifying SpLRA genes by geno-mic PCR using oligonucleotide primers designed from the Sphingomonas sp SKA58 genome sequence (see below) Unless otherwise noted, Sphingomonas sp hereafter indicates strain NBRC 101715
Functional expression of SpLRA genes in E coli Among the five SpLRA gene products, SpLRA3, SpLRA5, and SpLRA6 were successfully expressed in
E coli cells as (His)6-tagged enzymes The recombi-nant enzymes were purified to homogeneity using a nickel-chelating affinity column and then gel filtration chromatography (Fig 3B) Western blot analysis with
Trang 5an antibody against tag confirmed the
(His)6-tag at the N-terminal
L-Rhamnose-1-dehydrogenase (SpLRA1)
To initially estimate the physiological role of the LRA
gene cluster, we first attempted to biochemically
char-acterize a dehydrogenase with l-rhamnose in
Sphingo-monas sp When compared with nutrient medium
(0.035 unitÆmg)1 protein), approximate 9.7-fold higher
activity of NADP+-dependent dehydrogenation with
l-rhamnose was found in the cell-free extract from
Sphingomonassp cells grown on l-rhamnose (0.34 uni-tÆmg)1protein) Similar induction of NAD+-dependent activity by l-rhamnose was also found, although the specific activity was slightly lower than the NADP+ -dependent activity The l-rhamnose-1-dehydrogenase was (partially) purified by four chromatographic steps: there was NADP+-dependent and NAD+-dependent specific activity of 39.8 and 31.7 unitÆmg)1 protein, respectively (Fig 3A) A typical result of purification
is summarized in Table 1A During the purification procedure, the ratio of NADP+-linked and NAD+ -linked activity remained almost constant, suggesting the presence of only one protein as l-rhamnose-1-dehydrogenase Two major protein bands were found
on SDS⁄ PAGE gel (bands A and B, respectively, in Fig 3A) The N-terminal amino acid sequence up to
20 amino acids of band B, MKLLEGKTVLITGAST GIGR, was completely identical to that of SpLRA1, and the putative molecular mass of SpLRA1 (26.5 kDa) was similar to that of the native enzyme ( 28 kDa) (band A is described in the next section) Significant dehydrogenase activity was observed with
l-rhamnose, l-lyxose (75%) and l-mannose (8.4%) in the presence of NADP+ [values in parentheses are activity relative to that with l-rhamnose (100%)] The substrate specificity and dual coenzyme specificity between NAD+and NADP+(see above) were similar
to the same bacterial AvLRA1 (high concomitant activity for l-lyxose), compared with fungal PsLRA1 and DhLRA1 (strict NAD+-dependence) [12] These results indicated that SpLRA1 encodes NAD(P)+ -dependent l-rhamnose-1-dehydrogenase, and that the
91 65 48 37
28
M 1 2 3 kDa
kDa
195
91
48
37
28
1 2 3 4
20.5
Band A
Band B
Fig 3 SDS ⁄ PAGE (A) Purification of native L -rhamnose
dehydro-genase from Sphingomonas sp Lane 1: cell-free extract (100 lg).
Lane 2: HiPrep 16 ⁄ 10 Q FF (50 lg) Lane 3: HiPrep 16 ⁄ 10 Butyl FF
(20 lg) Lane 4: hydroxyapatite (20 lg) Lane 5: HiLoad 26 ⁄ 60
Superdex 200 pg (20 lg) M is marker protein Bands A and B
correspond to L -KDR 4-dehydrogenase and L
-rhamnose-1-dehydro-genase, respectively (see text) (B) Purification of (His)6-tagged
SpLRA3 (lane 1), SpLRA5 (lane 2), and SpLRA6 (lane 3) (each of
5 lg).
Table 1 Summary of concomitant purification of L-rhamnose-1-dehydrogenase (A) and L -KDR 4-dehydrogenase (B) from Sphingomonas sp.
Total activity (units)
Specific activity a
(unitsÆmg)1protein)
Yield (%) Purification fold
A
Step
Total protein (mg)
Total activity (units)
Specific activity (unitsÆmg)1protein)
Yield (%)
Purification fold B
a
NADP+-dependent activity.
Trang 6remaining LRA genes may also be related to
l-rham-nose metabolism
L-2-Keto-3-deoxyrhamnonate-(4)-dehydrogenase
(SpLRA5)
As described above, although SpLRA5 belongs to the
SDR family, together with SpLRA1, the enzyme
func-tion may be different from the dehydrogenafunc-tion with
l-rhamnose At present, this protein family contains
approximately 3000 primary structures and consists of
enzymes of several EC classes [20], and relatively high
degrees of similarity to SpLRA5 were found in several
(putative) reductases⁄ dehydrogenases (30–35%
identi-ties) Significant NAD+-dependent dehydrogenation
activity (0.22 unitÆmg)1 protein) of l-KDR was found
when Sphingomonas sp was grown on l-rhamnose, but
not when it was grown on nutrient medium
Further-more, no aldolase activity of l-KDR was induced by
l-rhamnose Surprisingly, the dehydrogenase for
l-KDR was concomitantly purified with
l-rhamnose-1-dehydrogenase: NAD+-dependent specific activity of
5.48 unitÆmg)1 protein was found (Table 1B) In the
(partially) purified sample, band A should correspond
to this enzyme (Fig 3A), and the N-terminal amino
acid sequence up to 19 amino acids was significantly
homologous with that of SpLRA5: (M)SVFAGRYA
GRXAIVTGGAS (underlined letters indicate the same
amino acids as SpLRA5; X, residue was not
deter-mined) On the other hand, the molecular mass of the
native enzyme estimated from SDS⁄ PAGE ( 29 kDa)
was slightly higher than the value estimated from the
putative amino acid sequence of SpLRA5 (25.7 kDa),
although this is not entirely clear These results
suggested that SpLRA5 plays a role as a novel
NAD+-dependent l-KDR dehydrogenase involved in
l-rhamnose metabolism in Sphingomonas sp
When the purified (His)6-tagged SpLRA5 was
incu-bated with each l-2-keto-3-deoxyacid-sugar in the
presence of NAD+, clear dehydrogenation activity
was detected by a spectrophotometric assay, and the
determined kinetic parameters are shown in Table 2
The kcat⁄ Km value with l-KDR was 81 min)1Æmm)1
in the range expected for the physiological substrate; this was 9.3-fold and 214-fold higher than those with 3-deoxylyxonate (l-KDL) and l-2-keto-3-deoxymannonate (l-KDM), respectively, this being mainly caused by the higher value of kcat l-Rhamnose was an inactive substrate, and no activity was found
in the presence of NADP+ when l-KDR was used
as a substrate To identify the hydrogen absorbed
by SpLRA5, an attempt was made to isolate the reaction product free of NAD+, protein, and buffer, but this proved to be unsuccessful, probably because of the unstable nature of the product Therefore, the reaction product of SpLRA5 was esti-mated by a coupling reaction with SpLRA6, as described below
The common active center of SDR enzymes consists
of a tightly conserved Ser-Tyr-Lys catalytic triad [20] Furthermore, the coenzyme-binding mode follows a classical ‘Rossmann fold’, in which a characteristic GXXXGX[G⁄ A] fingerprint motif exists These motifs are also conserved in SpLRA5: Ser143–Tyr156–Lys160 and Gly17-Gly-Ala-Ser-Gly-Leu-Gly23 (Fig 4A) These findings suggested that the fundamental catalytic mechanism and coenzyme recognition of SpLRA5 may
be similar to those in known SDR enzymes The recombinant SpLRA5 enzyme formed a homotetra-meric structure by itself, like other SDR proteins (data not shown) Therefore, it is likely that SpLRA1 is completely unnecessary to maintain the active form of SpLRA5, and that concomitant purification of SpLRA5 and SpLRA1 (Fig 3A) is due to the similar properties on the surface rather than the hetero-oligo-meric structure, although their sequence identity is only 29%
L-2,4-Diketo-3-deoxyrhamnonate hydrolase (SpLRA6)
SpLRA6 is a novel member of the MhpD family (COG0179), which is different from the protein fami-lies of LRA1–4 proteins (Fig 1) In HPLC analysis (Fig 5A), the retention time of the reaction product of SpLRA5 was almost the same as that of l-KDR
Table 2 Kinetic parameters of recombinant SpLRA5 protein Values are the means ± standard deviation, n = 3.
Substrate
Specific activity a
a
Under standard assay conditions as described in Experimental procedures.bTen different concentrations of substrate between 0.2 and
10 m M were used.
Trang 7( 10 min) Similar results were also observed with
l-KDL and l-KDM On the other hand, when
l-KDR was incubated with SpLRA5 and SpLRA6 in
the presence of NAD+, a novel peak with a later
retention time (13.1 min) appeared, identical to that of
l-lactate In the case of l-KDL, the peak corresponded
to glycolate Clearer results were obtained with
l-KDM: two peaks that differed from that of l-KDM ( 10 and 11.4 min) were observed, and were found
to be identical to those of pyruvate and (dl-)glycerate, respectively These results indicated that SpLRA6 cata-lyzes the hydrolysis of l-2,4-diketo-3-deoxyacid-sugar
C O O H
H H
H O H
H O H
C H 3
O
L -KDR L -KDF
C O O H
H H
H O H
H O H
C H 3
O
C O O H
H O H
H O H
H O H
H O H
H
H
O H
1
2
3
4
5
6
D -Gluconate
EC 1.1.1.215
D -2-Deoxygluconate
C O O H
H H
H O H
H O H
H O H
H
H
O H
C O O H
H H
H O H
H O H
H
H
O H
O
D -KDG
EC 1.1.1.126
EC 1.1.1.127 (KduD)
L -Rhamnose
C O O H
H O H
H O H
H O H
H O H
H
H
O H
C O O H
H H
H O H
H O H
H
H
O H
H
O H
H
O H
H
O H
H 3 C
H O
EC 1.1.1.264 (IdnD)
C O O H
H O H
H O H
H O H
H
H
H
O H
H O
L -Idonate
C O O H
H
H H
O
O H
O
H O H
C H 3
C O O H
H H
O
H O H
C H 3
O H
2 O
L -DKDR hydrolase (EC 3.7.1.-)
Pyruvate
L-Lactate L-DKDR
C O O H
H
H
O
H
H
O
H
C O O H
H
C O O H
H
H
O
H
H
H
C O O H
H
C O O H
H
H2O FAH (EC 3.7.1.2)
4-Fumarylacetoacetate
Fumarate
Acetoacetate
C O O H
O
H
H
H
H
C O O H
H
H
C O O H
O
H
C O O H
H
H
C O O H
H
H
CO2 HpcE (EC 4.1.1.68)
5-Oxopent-3-ene- 1,2,5-tricarboxylate
2-Oxohept-3- ene-1,7-dioate
C O O H
H H
C H 2 O H
H O H
O
C O O H
H H
C H O
H H
O
H2O
D-KDA dehydratase
C H 2
H
H
H O
C O O H
H O
H
H O
C O O H
C H 3
H2O MhpD (EC 4.2.1.80) 4-Hydroxy-2-
oxopentanoate
2-Hydroxy-2,4- pentadienoate
EC 1.1.1.69 EC 1.1.1.125
(KduD)
EC 1.1.1.173
D E E G
D E E G
L E E A
H E E V
D E E A
R E E A
N V E F Q
N L E E Q
D V A D E
N Y I D L
N W A D Q
L V G - I
p R 6
u E
d D
S
F
K
H c
A
F
M p
W 1 K
W 2 K
L 4 K
N 7 K
P 9 K
T 0 N
W 4 K
W 5 K
L 6 K
N 0 K
P 3 K
T 3 N
K - -
L - -
A N -
E - -
Q E V
R W I
W K K H T
W K K A T
L Q K Y G
N R K R G
P L K F -
T A N S G
P D M T T P V
P D I T T P V
D T L T T I P
P D I T T K L
P D L S T S S
T D I T A G M
W 3 K
W 3 K
L 4 K
N 8 K
P 4 K
T 2 N
R T
H G
- L
Y R
- L
Q V
A L A
S L A
S L A
F c
I n
K u
V S
V S
L S
S L G A Q H T T A
S L G E Q H T T A
A K G P A A S S A
M S A S K V N F Y V K A
I S Q
I S L
S
S
E A P I P T T G
F G I V S T S K
-
-
-
-
-
G 4 A
Q 4 A
D 4 V
D 4 T
L 4 T
R 4 S
V G S G G A
V G S G G A
V G A G G Q
I A G G G E
V G S G G T
I G D G G G
0
1
1
2
1
1
A
B
C
D
* * * *
Fig 4 (A) Partial sequence alignment between L -rhamnose-1-dehydrogenases from A vinelandii (AvLRA1) and Sphingomonas sp (SpLRA1, this study), L -KDR 4-dehydrogenase from Sphingomonas sp (SpLRA5, this study), L -KDF 4-dehydrogenase from X campestris (FucD), D -glu-conate 5-dehydrogenase from Gluconobacter oxydans (IdnO, CAA56322), and D -KDG 5-dehydrogenase from Erwinia chrysanthemi (KduD, CAA43989) Open and closed circles indicate NAD(H)+-dependent and NADP(H)+-dependent enzymes, respectively Asterisks, GXXXGX(G ⁄ A) coenzyme-binding motif of Rossmann fold; diamond, Ser-Tyr-Lys catalytic triad (B) Structurally analogous nonphosphorylated substrate to
L -KDR Each dehydrogenase acts at the gray-shadowed boxes Enzymes in boxes belong to the SDR protein family as described in (A) (C) Partial sequence alignment between L -DKDR hydrolases from Sphingomonas sp (SpLRA6, this study) and X campestris (FucE), D -KDA dehydratase from S solfataricus (KdaD; Protein Data Bank ID 2Q18), HpcE from E coli (1I7O), FAH from mouse (1QQJ), and MhpD from
E coli (1SV6) Circles, metal ion ligands; triangles, active sites The full (structure-based) sequence alignment is shown in Fig S2 (D) Schematic reactions of MhpD family enzymes Black triangles indicate cleavage sites of C–C bonds.
Trang 8to pyruvate and hydroxyl acid [physiologically,
l-2,4-diketo-3-deoxyrhamnonate (l-DKDR) hydrolase]
and that SpLRA5 should be assigned as ‘l-KDR
4-dehydrogenase’, which produces l-DKDR from
l-KDR
The MhpD family contains the archetypal MhpD
(EC 4.2.1.80) [21] fumarylacetoacetate hydrolase (FAH;
EC 3.7.1.2) [22],
5-oxopent-3-ene-1,2,5-tricarboxy-late decarboxylase⁄ 2-hydroxyhepta-2,4-diene-1,7-dioate
isomerase (EC 4.1.1.68) [23], and
d-2-keto-3-deoxyara-binonate (d-KDA) dehydratase [14,24] (Fig 4C; the full
sequence alignment is shown in Fig S2) Among them,
FAH catalyzes the hydrolytic cleavage of a C–C bond in
fumarylacetoacetate to yield fumarate and acetoacetate,
and the catalytic reaction is partially analogous to that
of l-DKDR hydrolase (Fig 4D) Furthermore, it is noteworthy that d-KDA dehydratase is involved in an archeal d-arabinose pathway that is (partially) analogous to the npED pathway and similar to the alter-native l-rhamnose pathway (E73) (Fig 1) MhpD family enzymes contain Ca2+ (5-oxopent-3-ene-1,2,5-tricarboxylate decarboxylase⁄ 2-hydroxyhepta-2,4-diene-1,7-dioate isomerase and FAH) or Mg2+ (d-KDA dehydratase) in the active center; these are coordinated with two highly conserved glutamates and one aspartate (Fig 4C) l-DKDR hydrolase also possesses a structur-ally equivalent metal ion-binding site, Glu119–Glu121– Asp150, whereas there are several significant variations
C O O H
H H
H O H
H O H
C H 3
O
C O O H
H H
O
H O H
C H 3
O
C O O H
H O H
C H 3 +
L -Lactate
C O O H
C H 3
O
L -KDR
Pyruvate
3-deoxyrhamnonate
C O O H
O
H H
H O H
C H 2 O H
C O O H
C + Glyocolate
L -KDL
C O O H
C H 3
O
H 2 O H
3-deoxylyxonate
C O O H
H H
C H 2 O H
O
O
C O O H
H H
H O H
H O H
C H 2 O H
O
C O O H
H H
O
H O H
C H 2 O H
O
C O O H
H O H
C H 2 O H
C O O H
C H 3
O + Glycerate
3-deoxymannonate
0
200
0
200
0
200
0
200
400
0
200
0
200
0
200
400
0
200
0
200
0
200
400
L -KDR + SpLRA5 + NAD+
L -KDR + SpLRA5 + NAD+ + SpLRA6
L -KDR
L -KDL + SpLRA5 + NAD+
L -KDL + SpLRA5 + NAD++ SpLRA6
L -KDM + SpLRA5 + NAD+
L -KDM + SpLRA5 + NAD+
+ SpLRA6
0
200
Pyruvate + Glycolate
8 9 10 11 12 13 14
Pyruvate + L -Lactate
Retention time (min)
0
200
Pyruvate + DL-Glycerate
A
B
Fig 5 (A) HPLC analysis of the reaction products from L -KDR, L -KDL and L -KDM formed by SpLRA5 and SpLRA6 Authentic pyruvate, L -lac-tate, glycolate and DL -glycerate were present at a concentration of 10 m M Arrows indicate the peak corresponding to pyruvate produced from L -KDM (B) Schematic of enzyme reaction products from L -KDR, L -KDL and L -KDM formed by L -KDR 4-dehydrogenase (LRA5) and
L -DKDR hydrolase (LRA6) Pyruvate is shown in the dashed box.
Trang 9in several amino acids in the (putative) active sites,
which may reflect their different catalytic reactions
L-Rhamnonate dehydratase (SpLRA3)
As AvLRA3 utilizes only l-rhamnonate, l-lyxonate
and l-mannonate as substrates efficiently [13], other
(dl-)2-keto-3-deoxyacid-sugars (and
2-diketo-3-deoxy-acid-sugars) in addition to l-KDR, l-KDL and
l-KDM are unavailable as substrates for SpLRA5 and
SpLRA6 Therefore, there is still a possibility that the
gene cluster of Sphingomonas sp is responsible for the
metabolism not only of l-rhamnose but also of other
sugars In this regard, SpLRA3 is more suitable for
estimating the physiological role of the gene cluster as
well as of SpLRA1, because a library was constructed
previously of 11 acid-sugars as potential substrates for
acid sugar dehydratases [12]
Therefore, recombinant (His)6-tagged SpLRA3 was
prepared using the same procedures as for SpLRA5
and SpLRA6 (Fig 3B) By semicarbazide endpoint
measurement, significant activity of SpLRA3 was
found with l-rhamnonate, l-lyxonate (128%), and
l-mannonate (89.2%) [values in parentheses are
rela-tive to the activity with l-rhamnonate (100%)]
Furthermore, SpLRA3 showed similar kinetic
para-meters with l-rhamnonate to those of AvLRA3 [12]
(Table 3), and the kcat⁄ Km value with l-rhamnonate
(177 min)1Æmm)1) was 26.2- and 59.2-fold higher than
those with l-lyxonate (6.75 min)1Æmm)1) and
l-manno-nate (2.99 min)1Æmm)1), respectively, mainly due to
significantly higher Km values (13.6- and 30.2-fold,
respectively) These results clearly suggested that
SpLRA3 should be assigned as an ‘l-rhamnonate
dehydratase’ and that the gene cluster of Sphingomonas
sp is related only to l-rhamnose metabolism
In vivo expression of LRA genes in an
L-rhamnose-defective E coli mutant
We here identified an alternative l-rhamnose pathway
in Sphingomonas sp (referred to as the
‘diketo-hydro-lase pathway’) that differs from the complete
analo-gous pathway to npED pathway (aldolase pathway) (Fig 2A) To estimate the physiological meaning of both pathways of l-rhamnose metabolism in vivo, genes LRA1–6 were introduced into plasmid vectors for multiple gene expression and transformed into an
l-rhamnose-defective E coli mutant, the KRX strain (Fig S1 and Table S1) In this study, the genes for AvLRA1, DhLRA2, AvLRA3, PsLRA4, AvLRA4, SpLRA5 and SpLRA6 were used, because of expres-sion level and⁄ or solubility problems with other LRA proteins in E coli cells (see Experimental procedures) Western blot analysis using (His)6-tag attached to the N-terminus of all LRA proteins revealed their func-tional expression in E coli cells grown on a nutrient medium supplemented with 0.2% (w⁄ v) l-rhamnose (Fig 6A) On the other hand, when the recombinant
E coli strains were cultivated in a minimal medium containing 2% (w⁄ v) l-rhamnose, the l-rhamnose-neg-ative phenotype was compensated for by introduction
of the genes LRA1–4 [1234(Ps) and Duet-1234(Av)] or LRA1–3, LRA5, and LRA6 (Duet-12356) (Fig 6B), suggesting significant physiological roles of both of the alternative l-rhamnose pathways in vivo Unexpectedly, the introduction of only LRA1 and LRA2 also led to slow growth of the cells (see Duet-12) Indeed, E coli possesses genes homologous to LRA3 and LRA4 (that encoding yfaW, 63.4% identity with AvLRA3; that encoding yfaU, 50.2% identity with AvLRA4) (Fig 2B), and their enzyme functions were recently assigned as l-rhamnonate dehydratase and l-KDR aldolase, respectively [25,26]
Discussion
As illustrated in Fig 1, there is significant phylogenetic mosaicism between the metabolic enzymes involved in sugar pathways analogous to the ED pathway: sugar dehydrogenase, lactone-sugar hydrolase (lactonase), acid-sugar dehydratase, and aldolase and dehydratase for (dl-)2-keto-3-deoxyacid-sugar One of the most interesting findings in this study was that two enzymes belonging to the same protein family (COG1028) are involved in a single sugar metabolic pathway: LRA1
Table 3 Kinetic parameters of recombinant SpLRA3 protein Values are the means ± standard deviation, n = 3.
Substrate
Specific activity a
kcat⁄ K m
(min)1Æm M )1)
L -Rhamnonate b 0.401 ± 0.03 (0.891 ± 0.058) c 0.121 ± 0.006 (0.115 ± 0.001) 21.3 ± 0.6 (43.1 ± 0.2) 177 ± 5 (375 ± 3)
a
Under standard assay conditions as described in Experimental procedures.bTen different concentrations of substrate between 0.02 and
1 m M were used c Values of AvLRA3 [12] d Six different concentrations of substrate between 0.5 and 5 m M were used.
Trang 10and LRA5 In the ‘recruitment model’ of enzyme
evo-lution proposed by Jensen [27], new enzymes evolve by
duplication and mutation of the same enzyme classes
from other pathways, leading to ‘catalytically
promis-cuous’ enzymes and ‘patchwork’-like metabolic
path-ways In case of the diketo-hydrolase pathway of
Sphingomonas sp., similar evolutionary processes have
occurred more than once in the same pathway
The ED pathway often plays a role as the metabolic
funnel of the npED pathway For example, in several
hyperthermophilic Archaea, d-2-keto-3-deoxygluconate
(d-KDG) and glycerate (produced from
d-glyceralde-hyde) are phosphorylated by specific kinases and
subsequently metabolized through the ED pathway: the
so-called ‘semi-phosphorylative ED pathway’ [28]
On the other hand, in several bacterial pathways,
d-gluconate and⁄ or d-KDG is produced from
d-2,5-di-keto-3-deoxygluconate (in pectin degradation [29]),
d-5-dehydrogluconate (in the l-idonate pathway [30]), and
d-2-deoxygluconate (in the 2-deoxyglucose pathway
[31]), and the (reverse) reactions, in particular the first
reaction, are analogous to that of SpLRA5 Therefore,
it may be reasonable to suppose that l-KDR
4-dehydro-genase is phylogenetically similar to the enzymes
cata-lyzing these reactions [29,32] (Fig 4A,B)
To the best of our knowledge, the conversion of
l-KDR into pyruvate and l-lactate via l-DKDR is a
novel metabolic fate in the known sugar pathways
Gerlt et al [33] recently identified a gene cluster related
to the alternative l-fucose metabolism of the bacterium
Xanthomonas campestris (E37–E41) (Fig 2B) Among the protein products of the five metabolic genes, FucB, FucC, FucD, and FucE are homologous to SpLRA2 (28% identity), SpLRA3 (30% identity), SpLRA5 (35% identity; see Fig 4A), and SpLRA6 (56% identity, see Fig 4D), respectively, suggesting that
l-DKDR is also produced from l-2-keto-3-deoxyfuco-nate (l-KDF) in this pathway On the other hand, as
l-fucose and l-fuconate are inactive substrates for SpLRA1 and SpLRA3, respectively (see Results), the LRAgene cluster should be related only to l-rhamnose metabolism of Sphingomonas sp but not l-fucose metabolism; in contrast, the archaeon Sulfolobus solfataricusmetabolizes both d-glucose and d-galactose promiscuously through the npED pathway [4,5]
l-Lactaldehyde is subsequently converted to l-lactate
by l-lactaldehyde dehydrogenase in the aldolase path-way [13], whereas the continuous reactions of l-KDR 4-dehydrogenase and l-DKDR hydrolase allow the metabolism of l-rhamnose into the same pyruvate and
l-lactate products without involvement of the physio-logically toxic aldehyde (Fig 2A), by which the diketo-hydrolase pathway may be more favorable than the aldolase pathway
Another interesting insight obtained in this study is that l-DKDR hydrolase belongs to the same MhpD protein family as d-KDA dehydratase (Fig 4C) As mentioned previously, there is another version of the npED pathway (type II in Fig 1), in which the
dl-2-keto-3-pentonate intermediate is converted to
50
30
M
2
1
2
3
1
2
3
4
1
3
4
2
1
3
5
2
3
1 5
2 6
1234(Ps) 1234(Av) kDa
E coli Duet strains
A
B
0 0.2 0.4 0.6 0.8
1 1.2 1.4
Days
1234(Av) 1234(Ps)
12356
12
0
Fig 6 Expression of LRA genes in an
L -rhamnose-defective mutant of E coli The
constructed recombinant E coli strains are
summarized in Table S3 (A) Western blot
analysis All LRA enzymes were
overexpres-sed as (His)6-tagged proteins in E coli cells
grown in a nutrient medium supplemented
with L -rhamnose, and purified on an
Ni2+-chelating affinity column One hundred
micrograms of each of the purified proteins
was applied to 11% (w ⁄ v) gel M indicates
marker protein (B) Growth in M9 minimal
liquid medium supplemented with 2% (w ⁄ v)
L -rhamnose.