Physiological characterization using recombinant enzymes revealed that, of the tested aldehyde substrates, l-lactaldehyde is the best substrate for both PsLADH and AvLADH, and that PsLAD
Trang 1alternative L-rhamnose pathway
Seiya Watanabe1,2,3, Sommani Piyanart1and Keisuke Makino1,2,3,4
1 Institute of Advanced Energy, Kyoto University, Japan
2 New Energy and Industrial Technology Development Organization, Kyoto, Japan
3 CREST, JST (Japan Science and Technology Agency), Japan
4 Innovative Collaboration Center, Kyoto University, Japan
l-Rhamnose (l-6-deoxymannose) is a constituent of
glycolipids and glycosides, such as plant pigments,
pectic polysaccharides, gums and biosurfactants, and
can be utilized as the sole carbon and energy source by
most bacteria, including Escherichia coli and Salmonella typhimurium In this pathway, l-rhamnose is converted into dihydroxyacetone phosphate and l-lactaldehyde via l-rhamnulose and l-rhamnulose l-phosphate by the
Keywords
Azotobacter vinelandii; L -lactaldehyde
dehydrogenase; L -rhamnose metabolism;
molecular evolution; Pichia stipitis
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 8 July 2008, revised 9 August
2008, accepted 15 August 2008)
doi:10.1111/j.1742-4658.2008.06645.x
Fungal Pichia stipitis and bacterial Azotobacter vinelandii possess an alter-native pathway of l-rhamnose metabolism, which is different from the known bacterial pathway In a previous study (Watanabe S, Saimura M
& Makino K (2008) Eukaryotic and bacterial gene clusters related to an alternative pathway of non-phosphorylated l-rhamnose metabolism
J Biol Chem 283, 20372–20382), we identified and characterized the gene clusters encoding the four metabolic enzymes [l-rhamnose 1-dehydrogenase (LRA1), l-rhamnono-c-lactonase (LRA2), l-rhamnonate dehydratase (LRA3) and l-2-keto-3-deoxyrhamnonate aldolase (LRA4)] In the known and alternative l-rhamnose pathways, l-lactaldehyde is commonly pro-duced from l-2-keto-3-deoxyrhamnonate and l-rhamnulose 1-phosphate by each specific aldolase, respectively To estimate the metabolic fate of l-lact-aldehyde in fungi, we purified l-lactl-lact-aldehyde dehydrogenase (LADH) from
P stipitis cells l-rhamnose-grown to homogeneity, and identified the gene encoding this enzyme (PsLADH) by matrix-assisted laser desorption ioniza-tion-quadruple ion trap-time of flight mass spectrometry In contrast, LADH of A vinelandii (AvLADH) was clustered with the LRA1–4 gene on the genome Physiological characterization using recombinant enzymes revealed that, of the tested aldehyde substrates, l-lactaldehyde is the best substrate for both PsLADH and AvLADH, and that PsLADH shows broad substrate specificity and relaxed coenzyme specificity compared with AvLADH In the phylogenetic tree of the aldehyde dehydrogenase super-family, PsLADH is poorly related to the known bacterial LADHs, includ-ing that of Escherichia coli (EcLADH) However, despite its involvement in different l-rhamnose metabolism, AvLADH belongs to the same subfamily
as EcLADH This suggests that the substrate specificities for l-lactaldehyde between fungal and bacterial LADHs have been acquired independently
Abbreviations
ALDH, aldehyde dehydrogenase; AvLADH, Azotobacter vinelandii LADH; EcLADH, Escherichia coli LADH; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; LADH, L -lactaldehyde dehydrogenase; LAR, L -lactaldehyde reductase; 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; MjLADH, Methanocaldococcus jannaschii LADH; PsLADH, Pichia stipitis LADH.
Trang 2sequential action of l-rhamnose isomerase (RhaA,
EC 5.3.1.14), rhamnulokinase (RhaB, EC 2.7.1.5) and
l-rhamnulose l-phosphate aldolase (RhaD, EC 4.1.2.19)
(Fig 1A) Most fungi, including Saccharomyces
cerevi-siae, cannot grow on d-xylose, l-arabinose and
l-rhamnose as the sole carbon source [1] However, Pichia stipitis possesses the ability to metabolize these sugars through alternative pathways different from
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 -KDR aldolase ( LRA4, EC 4.2.1.-)
ATP ADP
L -Rhamnose isomerase
(RhaA, EC 5.3.1.14)
L -Rhamnulokinase
(RhaB, EC 2.7.1.5)
L -Rhamnulose 1-P aldolase
(RhaD, EC 4.1.2.19)
Dihydroxyacetone-P
RhaD RhaA RhaB RhaS RhaR RhaT AAC76884 AAC76885 AAC76886 AAC76887 AAC76888 AAC76889
E coli
P stipitis
( L -Rhamnose:H + symporter) EAM07803 EAM07804 EAM07805 EAM07806 EAM07807 EAM07808 EAM07809 EAM07810
(Sugar transporter) (Sugar channel)
A vinelandii
ABN68602 ABN68405 ABN68404 ABN68603
AAC74497
Methylglyoxal
NADPH NADP + Glutathione
S-Lactoyl glutathione
NAD(P) +
NAD(P)H Lactate
Glutathione
NAD + NADH Pyruvate
Dihydroxyacetone-P
NADH NAD +
1,2-Propanediol P
NAD + NADH
L -Lactaldehyde dehydrogenase ( LADH, EC 1.2.1.22)
Lactaldehyde:propanediol oxidoreductase ( EC 1.1.1.77(55))
AAC75841 AAC75842 AAC75843 AAC75844 AAC75845 AAC75846
OH
H H
HO OH
H H O
H3C HO H H
HO OH
H H O
H3C HO H
O
CH3 H OH H OH OH H OH H HOOC
CH 3
H OH H OH H H
O HOOC
3
CH H OH OHC CH3
O HOOC
L -Rhamnose D -Xylose L -Arabinose
OH H
H HOH2C
H OH
HO H O
OH H
HOH2C H
H OH
HO H O
HOH2C OH H
H
OH
CH2OPO3 2-O
A
C
D
B
Fig 1 (A) Known bacterial L -rhamnose pathway (B) Novel non-phosphorylating L -rhamnose pathway In addition to L -rhamnose, Pichia stipi-tis (but not Saccharomyces cerevisiae) can metabolize D -xylose and L -arabinose to yield a common phosphorylated end-product, xylulose 5-phosphate (C) Schematic gene clusters related to L -rhamnose metabolism Chr 8 and Chr 2 in P stipitis indicates chromosome number Homologous genes are indicated in the same colour Fungal and bacterial LRA4 enzymes are not related evolutionally [3] LADH enzymes of
P stipitis and Azotobacter vinelandii (orange) were characterized in this study L -Fucose is converted to pyruvate and L -lactaldehyde through the analogous pathway to L -rhamnose, and metabolic genes, including FucO, are also clustered on the Escherichia coli genome (D) Metabolic network around L -lactaldehyde In this study, we focused on LADH (black line).
Trang 3the well-known bacterial pathways Although both
d-xylose and l-arabinose are converted into a
com-mon end-product, xylulose 5-phosphate, as in the
bacterial pathway, it is believed that l-rhamnose is
metabolized via non-phosphorylated intermediates
(Fig 1B) [2] In this pathway, l-rhamnose is oxidized
to l-rhamnono-c-lactone by NAD(P)+-dependent
dehydrogenase The lactone is cleaved by a lactonase
to l-rhamnonate, followed by a dehydration reaction
forming l-2-keto-3-deoxyrhamnonate (l-KDR) The
last step is the aldol cleavage of l-KDR to pyruvate
and l-lactaldehyde We are in the process of
enzy-matically and genetically characterizing the
alterna-tive l-rhamnose pathway of P stipitis, and recently
identified four metabolic enzymes: l-rhamnose
1-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-KDR
aldol-ase (LRA4) [3] The LRA1–4 genes were clustered on
the P stipitis genome (Fig 1C), and the homologous
gene cluster was found on the genomes of many
fungi as well as several bacteria, including
Azoto-bacter vinelandii
In the known and alternative l-rhamnose pathways,
the final reaction step is catalysed by each specific
aldolase to commonly yield l-lactaldehyde as one of the
products There are two known enzymes for
l-lact-aldehyde in bacteria (Fig 1D) The first is oxidation
by NAD+-dependent l-lactaldehyde dehydrogenase
(EC 1.2.1.22, LADH) to produce l-lactate [4–6] In
E coli, the enzyme is commonly responsible for both
l-rhamnose and l-fucose metabolism, and is also
iden-tical to the glycolaldehyde dehydrogenase (EC 1.2.1.21)
involved in ethylene glycol metabolism and glyoxylate
biosynthesis [4,5] Under anaerobic conditions,
l-lactal-dehyde is reduced by NADH-dependent l-lactall-lactal-dehyde
reductase (LAR, EC 1.1.1.77) and the
l-1,2-propane-diol obtained is excreted in the medium In an E coli
mutant that can grow on l-1,2-propanediol as a sole
carbon source, LAR also functions as l-1,2-propanediol
dehydrogenase, so-called ‘lactaldehyde : propanediol
oxidoreductase’ [7] In contrast with bacteria, the
correct physiological role of l-lactaldehyde and related
enzymes in fungi has not yet been clarified Chen et al
[8] reported that the Gre2 (YOL151W) gene from
S cerevisiae encodes a NADPH-dependent
methyl-glyoxal reductase (EC 1.1.1.283) catalysing the
reduc-tion of methylglyoxal to d- and⁄ or l-lactaldehyde
Furthermore, Inoue et al [9] identified an aldehyde
dehydrogenase (ALDH) with specificity for
l-lact-aldehyde enzymatically but not genetically However, it
is well known that a toxic methylglyoxal is neutralized
to lactate via lactoylglutathione (but not l-lactaldehyde)
by glyoxalase I (EC 4.4.1.5, YML004C) and gly-oxalase II (EC 3.1.2.5, YDR272W)
In this regard, the alternative l-rhamnose pathway
is the significant physiological origin of l-lactaldehyde
in fungi In this study, we first identified a fungal LADH from P stipitis Furthermore, phylogenetic comparison with the LADH of A vinelandii revealed that the same alternative l-rhamnose pathways appeared by convergent evolution between fungi and bacteria
Results Metabolic fate ofL-lactaldehyde in P stipitis When compared with d-glucose medium, approxi-mately 30-fold higher NAD+-dependent dehydroge-nase activity for l-lactaldehyde was observed in the cell-free extract from P stipitis cells grown on l-rham-nose as the sole carbon source (Fig 2A) Similar results were observed when d-lactaldehyde was used as
a substrate instead of l-lactaldehyde In Zymogram staining analysis, active bands of NAD+-dependent dehydrogenases for l-lactaldehyde and d-lactaldehyde appeared in the same position (Fig 2B), and no active
Band A Band B
NAD
B
0.5 0.4 0.3 0.2 0.1 0 0.06 0.04 0.02 0
G R G R G R G R
–1 protein)
P stipitis
A vinelandii
PsLADH
PsALDH*
AvLADH
Fig 2 Translational and transcriptional regulation of LADH
Pichi-a stipitis Pichi-and AzotobPichi-acter vinelPichi-andii cells were cultured in synthetic medium containing D -glucose (G) or L -rhamnose (R) (2%, w ⁄ v) (A) NAD + - and NADP + -dependent dehydrogenase activity for L -lact-aldehyde (L) or D -lactaldehyde (D) in the cell-free extract Values are the means ± SD, n = 3 (B) Zymogram staining Fifty micrograms
of the cell-free extract were applied to a 6% (w ⁄ v) non-denaturing PAGE gel After electrophoresis, the gel was soaked in staining solution in the presence of 10 m M L - or D -lactaldehyde and 10 m M
NAD + (C) Transcriptional effect of carbon source on PsLADH, PsALDH* and AvLADH genes Total RNAs (4 lg per lane) were isolated from microorganism cells grown on the indicated carbon sources.
Trang 4band was observed in the presence of NADP+ (data
not shown), suggesting that the l-rhamnose-inducible
NAD+-dependent (or preferring) dehydrogenase for
l-lactaldehyde and d-lactaldehyde seems to derive
from the same enzyme, and that NADP+-dependent
activity may be derived from the concomitant activity
of other constitutively expressed ALDH(s) Under
anaerobic conditions, P stipitis could metabolize
l-rhamnose (data not shown) These results indicate
that the metabolic fate of l-lactaldehyde derived from
the alternative l-rhamnose pathway in P stipitis is
dehydrogenation by LADH
Purification of LADH from P stipitis (PsLADH)
PsLADH was purified from P stipitis cells grown on
l-rhamnose as a sole carbon source in four
chromato-graphic steps (Fig 3A) During the purification
proce-dure, the ratio of NAD+- to NADP+-linked activity
remained almost constant (2.2–3.0), suggesting the
presence of only one protein as LADH The purified
enzyme exhibited a clear preference for NAD+ over
NADP+, with NAD+- and NADP+-dependent
spe-cific activities of 6.85 and 2.26 unitsÆ(mg protein))1,
respectively SDS-PAGE revealed only one subunit
with an apparent Mr value of 55 kDa As it was
impossible to determine the N-terminal sequence
because of blocking, the peptide mass fingerprinting of
trypsin-digested fragments was alternatively performed
by MALDI-TOF MS, and LADH was identified as a
protein annotated as a putative ALDH of P stipitis CBS 6054 (ABN64318): 63% sequence coverage (Table S1) This protein consisted of a polypeptide of
495 amino acids with a calculated Mrof 53 488.85 Da, comparable with that of the purified LADH deter-mined by SDS-PAGE
For the known dehydrogenases for l-lactaldehyde, the reaction product of the enzymes from E coli [4,5], Methanocaldococcus jannaschii [10] and S cerevisiae [9]
is l-lactate (EC 1.2.1.22), whereas that from rat liver is methylglyoxal (EC 1.1.1.78) [11] In HPLC analysis, the retention time of the reaction product for PsLADH (13.32 min) was almost the same as that
of l-lactate (13.35 min), but not methylglyoxal (12.36 min); therefore, the enzyme catalyses the NAD(P)+-linked oxidation of l-lactaldehyde into
l-lactate The amino acid sequence of PsLADH was most closely related to E coli LADH (EcLADH) of the ALDH-like proteins on the P stipitis genome (34.5% identity), whereas the protein annotated as a putative mitochondrial ALDH (ABN68636) also showed similar homology to EcLADH (32.2% identity), indicating the possibility that the latter is an LADH isozyme (referred
to as PsALDH*); therefore, both enzymes were expressed in E coli cells (see below)
Candidate of LADH gene from A vinelandii
As described in the Introduction, we have previously identified the gene cluster related to the alternative
l-rhamnose pathway of A vinelandii [3] The LRA1–4 genes are clustered together with putative sugar trans-porters and the ALDH gene (EAM07810) (Fig 1C) This ALDH showed highest sequential similarity to EcLADH (61.7% identity) of all the putative ALDHs
in the A vinelandii genome, indicating that the protein may function as LADH (referred to as AvLADH) Two active bands corresponding to NAD+-dependent LADH were found in Zymogram staining analysis using the cell-free extract prepared from A vinelandii cells grown on l-rhamnose: strict l-rhamnose-inducible enzyme with l-lactaldehyde specificity (band A); mod-erate l-rhamnose-inducible enzyme that utilizes both
d- and l-lactaldehyde (band B) (Fig 2B) Subsequent characterization revealed that ALDH with EAM07810 may correspond to band A, a major LADH in l-rham-nose-grown cells (see below)
Functional expression of LADH in E coli PsLADH, PsALDH* and AvLADH genes were overex-pressed in E coli cells as a His6-tagged enzyme and purified homogeneously with a nickel-chelating affinity
M
19.5 kDa
119 kDa
91 kDa
65 kDa
48 kDa
37 kDa
28 kDa
Fig 3 (A) SDS-PAGE purification of native PsLADH in 10% (w ⁄ v) gel.
Lane 1, cell-free extracts (50 lg); lane 2, HiPrep 16 ⁄ 10 Q FF (50 lg);
lane 3, HiLoad 16 ⁄ 60 Superdex 200 pg (20 lg); lane 4, CHT
Ceramic Hydroxyapatite (20 lg); lane 5, Blue Sepharose Fast Flow
(10 lg) (B) SDS-PAGE of native and His6-tagged recombinant
enzymes Lane 1, native PsLADH; lane 2, His6-tagged PsLADH;
lane 3, His6-tagged PsALDH*; lane 4, His6-tagged AvLADH Ten
micrograms of the purified enzyme were applied Bottom panel:
immunoblot analysis using anti-His6-tag IgG One microgram of
the purified enzyme was applied.
Trang 5column (Fig 3B) Western blot analysis with
anti-His6-tag IgG confirmed the His6 tag in the enzyme
(bottom panel in Fig 3B)
Substrate specificity
Generally, ALDHs show relatively broad substrate
specificity in addition to the physiological substrate;
therefore, various aldehydes, including l-lactaldehyde,
were tested as substrates for dehydrogenation by purified proteins in the presence of NAD+, and the activity values for the tested aldehydes relative to
l-lactaldehyde are summarized in Table 1 l-Lactalde-hyde was the best substrate for PsLADH, and the specific activity [6.95 unitsÆ(mg protein))1] was compa-rable with that of native enzyme [6.85 unitsÆ(mg pro-tein))1] Only five other aldehydes showed more than 50% activity relative to l-lactaldehyde The significant utilization of d-lactaldehyde conformed to the preli-minary Zymogram staining analysis using the cell-free extract (Fig 2B) By contrast, PsALDH* utilized C2, C3 and C4 aldehydes more efficiently than l-lactalde-hyde, and most of the remaining aldehydes were also good substrates at varying rates up to about one-half the rate with l-lactaldehyde Overall, the specificity for
l-lactaldehyde of PsLADH was significantly higher than that of PsALDH*, conforming to the physiologi-cal role as a LADH involved in the alternative l-rham-nose pathway Comparable dehydrogenase activity of AvLADH with PsLADH was found only for l-lactal-dehyde and glycolall-lactal-dehyde, and activities with d-lactal-dehyde and C7 ald-lactal-dehyde were only 10% less than those with l-lactaldehyde: band A in Zymogram stain-ing may correspond to AvLADH (Fig 2B) These results suggest that the enzyme should be assigned to LADH, as expected from the sequential similarity to EcLADH
Kinetic analysis EcLADH functions as a glycolaldehyde dehydro-genase involved in ethylene glycol metabolism and glyoxylate biosynthesis [4,5] PsLADH, PsALDH*
Table 1 Substrate specificity of PsLADH, PsALDH* and AvLADH.
Substrate a
Relative activity (%) b
a The assay was performed with standard assay solution containing
10% (v ⁄ v) ethanol, 1 m M aldehyde and 1.5 m M NAD+using purified
His6-tagged recombinant enzymes b Relative values were
expressed as a percentage of the values obtained in L -lactaldehyde.
Table 2 Kinetic parameters of PsLADH, PsALDH*, AvLADH and EcLADH.
Enzyme Substrate Coenzyme Specific activity [unitÆ(mg protein))1]a Km (l M ) kcat (min)1) kcat ⁄ Km (min)1Æl M )1)
a Under standard assay conditions in Experimental procedures b Eight different concentrations of aldehyde between 2 and 100 l M were used c Eight different concentrations of glycolaldehyde between 10 and 100 l M were used d Calculation from data in [5].
Trang 6and AvLADH also utilize glycolaldehyde efficiently as a
substrate (Table 1); therefore, these enzymes were
sub-jected to further kinetic analysis with l-lactaldehyde,
d-lactaldehyde and glycolaldehyde, and the parameters
determined are listed in Table 2 The catalytic efficiency
(kcat⁄ Km) with l-lactaldehyde of PsLADH in the
pres-ence of NAD+(32.4 min)1Ælm)1) was 17.2-fold higher
than that of PsALDH* (1.88 min)1Ælm)1), caused by
both higher Km and lower kcat values However, by
contrast with l-lactaldehyde, the two fungal enzymes
possessed similar kcat⁄ Km values with d-lactaldehyde,
but their values with glycolaldehyde were significantly
lower, mainly caused by decreased kcat values When
NADP+ was used as a coenzyme, the kcat⁄ Km value
with l-lactaldehyde of PsALDH* decreased 15.8-fold
compared with that in the presence of NAD+because
of a decreased kcat value, whereas that of PsLADH
decreased only 1.6-fold These results suggest that
PsLADH possesses a stricter substrate specificity for
l-lactaldehyde and a more relaxed coenzyme specificity
than does PsALDH* Furthermore, the PsLADH gene
was significantly induced by l-rhamnose in P stipitis
cells, but the PsALDH* gene was not (Fig 2C) These
results strongly suggest the physiological function of
PsLADH in the alternative l-rhamnose metabolism
The kcat⁄ Km value with l-lactaldehyde of AvLADH
(15.6 min)1Ælm)1) in the presence of NAD+ was
55.5-fold higher than that with d-lactaldehyde
(0.281 min)1Ælm)1), and no activity was observed in
the presence of NADP+, in contrast with fungal
enzymes The kinetic parameters of l-lactaldehyde and
glycolaldehyde were similar to those of EcLADH [5]
Furthermore, the AvLADH gene was up-regulated
dur-ing growth on l-rhamnose (Fig 2C) As the activities
of LRA1–4 proteins were also significantly induced by
l-rhamnose-grown A vinelandii cells (data not shown),
the gene cluster containing LRA1–4 and AvLADH
genes may be strictly regulated by l-rhamnose as a
single transcriptional unit (Fig 1C)
Amino acid sequence analysis of LADH
In the phylogenetic tree of the ALDH superfamily,
PsLADH and PsALDH* fall into the fungal ALDH
subfamily, one of the 14 ALDH subfamilies compiled
by Perozich et al [12] (Fig 4), confirming the
micro-organism source The fungal ALDH subfamily belongs
to the Class 1⁄ 2 branch of ALDHs, which consists of
tetrameric ALDH subfamilies with variable substrate
specificity, as well as two P stipitis enzymes (Table 1)
In S cerevisiae, there is biochemical evidence of two
types of ALDH [13,14] The mitochondrial ALDHs,
ScALDH4 and ScALDH5, show dual coenzyme
speci-ficity between NAD+ and NADP+ and are activated
by K+ The cytosolic ALDHs, ScALDH2, ScALDH3 and ScALDH6, are specific to NADP+; only ALDH6
is activated by Mg2+ Higher degrees of similarity to PsLADH (probably cytosolic enzyme because of no mitochondrial leader sequence) were found in the mitochondrial ALDHs of S cerevisiae, confirming the enzyme properties, including coenzyme specificity Indeed, the activity of PsLADH is also absolutely dependent on K+ (data not shown) However, PsALDH* (cytosolic enzyme as well as PsLADH) is more closely related than PsLADH to cytosolic ALDHs, indicating that this enzyme may be assigned
as an acetaldehyde dehydrogenase rather than LADH, based on substrate specificity (Table 1) A branch of AvLADH and EcLADH was located on the root of the non-phosphorylating glyceraldehyde 3-phosphate dehydrogenase (GAPDH, EC 1.2.1.9.) subfamily in the Class 3 branch, consisting of substrate-specific ALDH subfamilies (Fig 4), confirming their enzyme proper-ties of high specificity with l-lactaldehyde (Tables 1 and 2) (dl-)Lactaldehyde dehydrogenase of archaeal
M jannaschii (MjLADH) is also a member of this subfamily, and is involved in the production of lactate for coenzyme F420biosynthesis [10]
Discussion
In this study, we have identified the LADHs involved
in the alternative l-rhamnose pathways of fungi and bacteria In particular, although fungi possess multiple ALDH genes, only one physiological substrate, acetal-dehyde, has been identified in fermentation and⁄ or growth on ethanol To our knowledge, this is the second report of fungal ALDH as an aldehyde substrate in addition to acetaldehyde; the other stated that ScALDH2 and ScALDH3 play a role as 3-amino-propionaldehyde dehydrogenases in pantothenic acid (vitamin B5) and coenzyme A biosynthesis [15]
Enzyme catalysis of LADH Hempel et al [16] proposed several characteristic con-served regions containing almost all active amino acid residues in ALDHs In particular, glutamate in the motif of LELGGKSP participates as a general base for the activation of catalytic cysteine and deacylation
of the enzyme, and cysteine in the motif of FXNXGQXCIA (where X is any amino acid) acts as a nucleophile These motifs are also conserved in PsLADH and AvLADH with a few modifications (Fig 5), indicating that the overall structure and fundamental catalytic mechanism may be similar to
Trang 7those in known ALDHs Based on structural studies of
other ALDHs, amino acid residues at equivalent
positions of 190 and 193 in PsLADH are involved in the
distinction between NAD+and NADP+ The
structur-ally equivalent lysine residue to Lys190 is conserved in
all ALDHs, and is unlikely to influence directly
coen-zyme specificity A glutamate residue at an equivalent
position to 193 interacts with 2¢- and 3¢-hydroxyl groups
of the ribose of the adenine moiety in strict NAD+ -preferring enzymes, such as EcLADH, AvLADH and PsALDH* However, the structurally equivalent gluta-mate is found not only in PsLADH with significant NADP+-dependent activity, but also in NADP+ -preferring ScALDH4 and ScALDH5 However, it is
Fig 5 Partial alignment of amino acid sequences around several active sites Open and filled circles indicate NAD + - and NADP + -dependent enzymes ScALDH4 (grey circle) utilizes both NAD+and NADP+as a coenzyme Grey-shaded letters are highly conserved In the crystal structure of EcLADH (PDB ID, 2IMP), open and filled stars indicate amino acid residues bound to L -lactate and 2¢- and 3¢-hydroxyl groups of NADH, respectively The catalytic glutamate and cysteine residues are indicated by grey stars.
Class 1
Class 2
Fungal ALDH
FTDH HMSALDH
Group X
BALDH
SSALDH
GAPDH
Aromatic ALDH
MMSALDH Turgor ALDH
GGSALDH
Class 3 ALDH
Class 1/2 branch Class 3 branch
PsALDH* ScALDH3 ScALDH2 ScALDH6
PsLADH ScALDH5 ScALDH4
Pichia angusta
Alternaria alternata Cladosporium herbarum Aspergillus nidulans Aspergillus niger Ustilago maydis
EcLADH AvLADH MjLADH
Fig 4 The overall phylogenetic tree of
known ALDHs, including LADHs Sequence
names and references of ALDHs are
avail-able on the ALDH website described in
Experimental procedures The three
enzymes in the boxes were characterized in
this study.
Trang 8known that ALDH isozyme B of E coli (AldB, 33%
identity to EcLADH) is a strict NADP+-dependent
enzyme and possesses the structural equivalent arginine
residue at this position (Arg197) [17] When compared
with the wild-type enzyme, the R197E mutant shows
10% NADP+-dependent activity, together with no
detection of NAD+-dependent activity In PsLADH
and PsALDH*, each E193R mutant expressed a
simi-lar level to the wild-type enzyme in E coli cells as
an inclusion body; we did not perform further
enzy-matic characterization (data not shown) This indicates
that, although the glutamate residue in PsLADH (and
fungal ALDHs) should play a role in coenzyme
bind-ing and⁄ or structural maintenance, other amino acid
residues may also influence coenzyme specificity
Inoue et al [9] purified a NAD+-dependent
dehydro-genase for l-lactaldehyde from S cerevisiae cells
cul-tured in a nutrient medium Although the enzyme has
not yet been characterized genetically, the molecular
structures (monomeric form consisting of the subunit
with Mrof 40 kDa) are clearly different from those of
general ALDH enzymes, including fungal ALDHs
(tetrameric or dimeric form consisting of the subunit
with Mr of 50–55 kDa, see Fig 3) Furthermore, the
activity with d-lactaldehyde is only 0.2% of that with
l-lactaldehyde, and acetaldehyde, dl-glyceraldehyde
and propionaldehyde are inactive substrates, in contrast
with PsLADH (Table 1); acetaldehyde is a common
active substrate for the known ScALDH2–6 Therefore,
although the genetic background and physiological
functions of LADH in S cerevisiae have not been
eluci-dated so far, it has been reported recently that the Gre2
(YOL151w) gene encodes methylglyoxal reductase,
related to the detoxification of methylglyoxal [8], in
which the LADH(-like) enzyme may also be involved
Convergent evolution of LADHs in fungi
and bacteria
In the phylogenetic tree, substrate-specific ALDHs
have a tendency to belong to subfamilies in the Class 3
branch, whereas ALDH families with broad substrate
specificity are more often found in the Class 1⁄ 2
branch (Fig 4) PsLADH (and also PsALDH*) shows
significant activity for several aldehydes in addition
to l-lactaldehyde (Table 1), and the fungal ALDH
subfamily containing this enzyme belongs to the
Class 1⁄ 2 branch However, AvLADH, which shows
high specificity to l-lactaldehyde, is similar to the
GAPDH subfamily in the Class 3 branch It is
note-worthy that, although l-lactaldehyde is produced by
the same alternative pathway of l-rhamnose in
P stipitisand A vinelandii, their LADHs are classified
into different subfamilies, strongly suggesting that their substrate specificities have been acquired by ‘conver-gent evolution’ rather than divergence from a common ancestor Indeed, four ligands for the substrate (l-lac-tate) are not conserved between PsLADH and EcLADH (Fig 5) PsLADH seems to have evolved from an ancestor with broader substrate specificity, such as PsALDH*, because PsALDH* is located at the root of the fungal ALDH subfamily (Fig 4)
It is certain that AvLADH and EcLADH, which are involved in different pathways of the same l-rhamnose metabolism, evolved from a common ancestor EcLADH is responsible for not only l-rhamnose but also l-fucose metabolism [5], whereas the LRA1–4 proteins, components of the gene cluster containing the AvLADH gene (Fig 1C), show no significant activ-ity with l-fucose-related intermediates [3] Therefore, it
is probable that the gene cluster is involved in l-rham-nose metabolism only, but not l-fucose MjLADH is involved in different metabolism from l-rhamnose (coenzyme F420 biosynthesis) [10] and is similar to EcLADH and AvLADH phylogenetically (Fig 5) Although glyceraldehyde 3-phosphate is commonly
an inactive substrate for AvLADH (see Table 1), EcLADH and MjLADH, MjLADH is capable of utilizing several aldehydes, such as glycolaldehyde,
dl-glyceraldehyde, formaldehyde, acetaldehyde and propionaldehyde (the last three are inactive substrates for AvLADH and EcLADH) Furthermore, substrate-binding sites of bacterial LADHs are not completely conserved in MjLADH (Fig 5) These results suggest that substrate specificity for l-lactaldehyde has also been acquired for bacteria and Archaea independently, similar to fungi
Of the 14 subfamilies in the ALDH superfamily, some subfamilies, such as c-glutamyl semialdehyde dehydro-genase, methylmalonyl semialdehyde dehydrogenase and succinic semialdehyde dehydrogenase, include sequences from organisms ranging from bacteria to mammals (Fig 4) [12] In contrast, the fungal ALDH subfamily (consisting of only fungal sequences) appears
to have diverged much later in evolution, indicating that the acquisition of substrate specificity for l-lactaldehyde might have occurred after divergence between bacteria and eukaryotes (fungi) Four metabolic enzymes (genes) that convert l-rhamnose into pyruvate and l-lactalde-hyde are found on the genomes of several fungi, includ-ing P stipitis, Debaryomyces hansenii, Candida species and Aspergillus species [3], but not S cerevisiae, which is not capable of growth on l-rhamnose [1] Therefore, the acquisition of these l-rhamnose metabolic genes might have led to the appearance of LADH from a common ancestor of fungal ALDHs under evolutionary pressure
Trang 9Experimental procedures
Microorganism strains, culture conditions and
preparation of cell-free extracts
Jef-fries (University of Wisconsin, Milwaukee, WI, USA)
National Institute of Technology and Evaluation (Chiba,
yeast nitrogen broth and Burk’s nitrogen-free medium
carbon source, respectively Usually, l-rhamnose was
sterili-zed separately by filtration and added to each medium The
grown cells were harvested by centrifugation at 30 000 g
for 20 min, washed with 20 mm potassium phosphate
(pH 7.5) containing 1 mm EDTA and 10 mm
use Fungal cells were suspended in Buffer A, homogenized
with an equal volume of glass beads (0.5 mm diameter,
Sigma, St Louis, MO, USA) for 30 min with appropriate
Mixer (AS ONE Co., Ltd., Osaka, Japan) and then
disrupted by sonication for 20 min with appropriate
Processor XL2020 (Misonix Incorporated, New York, NY,
USA) and then centrifuged
Enzyme activity assay
of Huff and Rudney [18] with a few modifications LADH
activity was assayed routinely in the direction of aldehyde
1 mm l-lactaldehyde, 1 mm EDTA and 10 mm
2-mercapto-ethanol in 66.7 mm potassium phosphate (pH 7.5) buffer
The reaction was started by the addition of 15 mm
1 mL In the case of water-insoluble aldehyde, 10 mm
substrates in ethanol (0.1 volume) were added to the
determined by the method of Lowry et al [19], with bovine
serum albumin as a standard
Zymogram staining analysis for LADH
Cell-free extracts were separated by non-denaturing PAGE
10 mL of staining solution [20] consisting of 100 mm
nitroblue tetrazolium, 0.06 mm phenazine methosulfate and
activity appeared as a dark band
Purification of native LADH from P stipitis
chromatography was carried out using an A¨KTA purifier system (Amersham Pharmacia Biotech, Little Chalfont,
Cell-free extracts prepared from l-rhamnose-grown P
equilibrated with Buffer A, and washed thoroughly with the same buffer The column was developed with 300 mL
of a linear gradient of 0–0.5 m NaCl in Buffer A Active fractions containing LADH were combined and
(Millipore, Bedford, MA, USA) at 18 000 g for approxi-mately 2 h The enzyme solution was loaded onto a column
fractions were pooled, concentrated and applied to a
Bio-Rad Laboratories, Hercules, CA, USA) equilibrated with Buffer A The column was washed thoroughly with the same buffer and developed with 150 mL of a linear gradient of 0–0.3 m potassium phosphate in Buffer A The fractions with high enzymatic activity were combined, con-centrated and loaded onto a column of Blue-Sepharose
equili-brated with Buffer A The column was washed with Buffer A containing 50 mm NaCl, and then the enzyme was eluted with Buffer A containing 1 m NaCl The elutant
until use
Determination of internal amino acid sequences
Purified PsLADH ( 50 lg) was separated by SDS-PAGE
performed according to a standard protocol [21] with a few modifications The peptide masses were analysed
ionization-quadruple ion trap-mass spectrometer (AXIMA QIT, Shimadzu, Kyoto, Japan) with 2,5-dihydroxybenzoic acid (Shimadzu GLC Ltd, Tokyo, Japan) as a matrix in positive ion mode
Identification of enzyme reaction product
HPLC analysis was performed using a Multi-Station
Trang 10PsLADH ( 1 mg) was added to a reaction mixture
acid was added to the samples (0.1 volume) to remove
7.8 mm, Bio-Rad) linked to an RID-8020 refractive index
Functional expression and purification of
His6-tagged proteins
Genomic DNA of P stipitis and A vinelandii was prepared
Japan) To introduce the restriction sites for BamHI and
PstI at the 5¢- and 3¢-termini of PsLADH, PsALDH* and
Japan) and appropriate primers (Table S2) Each amplified
DNA fragment was introduced into BamHI-PstI sites in
pQE-80L (Qiagen), a plasmid vector for conferring the
N-terminal His6 tag on expressed proteins E coli DH5a
turbidity of 0.6 at 600 nm in Super broth medium
iso-propyl thio-b-d-galactopyranoside, the culture was grown
for a further 6 h to induce the expression of His6-tagged
protein Cells were harvested and resuspended in Buffer B
(pH 8.0, 50 mm sodium phosphate containing 300 mm
NaCl, 10 mm 2-mercaptoethanol and 10 mm imidazole)
The cells were then disrupted by sonication, and the
solution was centrifuged The supernatant was loaded onto
a column of Ni-NTA Super Flow (Qiagen) equilibrated
Bio-Assist eZ system The column was washed with Buffer C
50 mm imidazole instead of 10 mm imidazole) The enzymes
were then eluted with Buffer C containing 250 mm
imid-azole instead of 50 mm imidimid-azole All His6-tagged enzymes
were used within 1 week in further experiments
Amino acid sequence alignment and
phylogenetic analysis
For phylogenetic analysis, 145 ALDH sequences were
obtained from a website devoted to ALDHs: http://www
psc.edu/biomed/pages/research/Col_HBN_ALDH.html [12]
The sequences were aligned using the program clustalw,
distributed by GenomeNet (Bioinformatics Center, Kyoto
University, Kyoto, Japan) (http://www.genome.jp) The
phylogenetic tree was produced using the treeview 1.6.1
program
Northern blot analysis
har-vested by centrifugation Total RNAs were prepared using
was carried out using a standard method The PCR prod-ucts of PsLADH, PsALDH* and AvLADH genes, amplified
by PCR using appropriate DNA primers, were labelled
(TaKaRa) and used as probes for hybridization
Acknowledgements This work was supported by a Grant-in-Aid for Young Scientists (B) (No 18760592) from the Ministry
of Education, Culture, Sports, Science and Technology
of Japan (to S.W.), by the Fermentation and Meta-bolism Research Foundation, by the Japan Bioindustry Association (to S.W.), by the Research Foundation from the Association for the Progress of New Chemis-try (to S.W.), by the New Energy and Industrial Tech-nology Development Organization (to S.W.) and by CREST, JST (to K.M.) We thank Dr T W Jeffries (University of Wisconsin, Milwaukee, WI, USA) for the gift of P stipitis CBS 6054 We are especially grateful to Dr M Yamada (Shimadzu Corporation, Kyoto, Japan) for his help with MALDI-TOF MS analysis
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