Metabolic fate of L-lactaldehyde derived from an
alternative L-rhamnose pathway
Seiya Watanabe
1,2,3
, Sommani Piyanart
1
and Keisuke Makino
1,2,3,4
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-lactaldehyde 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 LRA1–4gene 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.
FEBS Journal 275 (2008) 5139–5149 ª2008 The Authors Journal compilation ª2008 FEBS 5139
sequential 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-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
H2O
H2O
Pyruvate
L-Lactaldehyde
L-KDR aldolase
( LRA4, EC 4.2.1.-)
L-Rhamnose
L-Rhamnulose
L-Rhamnulose 1-P
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
ABN68602ABN68405ABN68404 ABN68603Chr 8 Chr 2 ABN64318
AAC74497
Methylglyoxal
NADPH
NADP+
Glutathione
L-Lactaldehyde
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))
FucO FucA FucP FucI FucK FucU
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
CH3
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
HOH
HO H
O
OH
H
HOH2C
H
HOH
HO H
O
HOH2C
OH
H
H
OH
CH2OPO32-
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).
L-Lactaldehyde dehydrogenase S. Watanabe et al.
5140 FEBS Journal 275 (2008) 5139–5149 ª2008 The Authors Journal compilation ª2008 FEBS
the 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 LRA1–4genes 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-lactaldehyde
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 of L-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
GR GR GR GR
LD LD
P. stipitis A. vinelandii
Band A
Band B
NAD
AC
B
+
NADP
+
0.5
0.4
0.3
0.2
0.1
0
0.06
0.04
0.02
0
GRGRGRGR
L D L D
Specific activity
(unit mg–1 protein)
P. stipitis
A. vinelandii
PsLADH
PsALDH*
AvLADH
Fig. 2. Translational and transcriptional regulation of LADH. Pichi-
a stipitis and Azotobacter vinelandii 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 mML-orD-lactaldehyde and 10 mM
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.
S. Watanabe et al. L-Lactaldehyde dehydrogenase
FEBS Journal 275 (2008) 5139–5149 ª2008 The Authors Journal compilation ª2008 FEBS 5141
band 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 M
r
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 M
r
of 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
1 2 345M
AB
M1234
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.
L-Lactaldehyde dehydrogenase S. Watanabe et al.
5142 FEBS Journal 275 (2008) 5139–5149 ª2008 The Authors Journal compilation ª2008 FEBS
column (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 glycolaldehyde, and activities with d-lactal-
dehyde and C7 aldehyde 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
PsLADH PsALDH* AvLADH
L-Lactaldehyde 100 100 100
D-Lactaldehyde 75 54 8.6
Formaldehyde (C1) 13 13 0
Acetaldehyde (C2) 67 309 0
Propionaldehyde (C3) 81 350 0
Butylaldehyde (C4) 39 175 0
Valeraldehyde (C5) 38 105 0
Hexylaldehyde (C6) 41 82 0
Heptylaldehyde (C7) 28 70 7.4
Octylaldehyde (C8) 22 57 0
Isobutylaldehyde 82 54 0
Glutaraldehyde 47 251 0
Glycolaldehyde 74 70 91
Benzaldehyde 27 23 0
Betaine aldehyde 13 15 0
Glyceraldehyde 30 27 0
Glyceraldehyde 3-phosphate 12 11 0
a
The assay was performed with standard assay solution containing
10% (v ⁄v) ethanol, 1 mMaldehyde and 1.5 mMNAD
+
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
K
m
(lM)k
cat
(min
)1
)k
cat
⁄K
m
(min
)1
ÆlM
)1
)
PsLADH L-Lactaldehyde
b
NAD
+
6.95 ± 0.10 42.8 ± 4.2 1390 ± 127 32.4 ± 0.2
NADP
+
1.65 ± 0.04 9.79 ± 0.74 195 ± 8 20.0 ± 0.3
D-Lactaldehyde
b
NAD
+
4.43 ± 0.05 52.9 ± 3.4 1460 ± 79 27.5 ± 0.3
Glycolaldehyde
c
NAD
+
8.94 ± 0.40 78.0 ± 1.6 469 ± 8 6.01 ± 0.03
PsALDH* L-Lactaldehyde
b
NAD
+
3.64 ± 0.10 350 ± 62 651 ± 112 1.88 ± 0.01
NADP
+
0.355 ± 0.007 131 ± 9 15.4 ± 0.7 0.119 ± 0.001
D-Lactaldehyde
b
NAD
+
2.10 ± 0.04 32.6 ± 5.6 89.5 ± 11.9 2.76 ± 0.10
Glycolaldehyde
c
NAD
+
4.28 ± 0.30 287 ± 12 137 ± 5 0.478 ± 0.003
AvLADH L-Lactaldehyde
b
NAD
+
17.2 ± 0.9 35.5 ± 0.8 554 ± 19 15.6 ± 0.2
D-Lactaldehyde
b
NAD
+
2.82 ± 0.04 167 ± 5 47.0 ± 1.5 0.281 ± 0.001
Glycolaldehyde
c
NAD
+
11.5 ± 0.4 274 ± 41 307 ± 44 1.12 ± 0.01
EcLADH
d
L-Lactaldehyde NAD
+
5.73 40 418 10.5
Glycolaldehyde NAD
+
14.7 380 993 2.61
a
Under standard assay conditions in Experimental procedures.
b
Eight different concentrations of aldehyde between 2 and 100 lMwere
used.
c
Eight different concentrations of glycolaldehyde between 10 and 100 lMwere used.
d
Calculation from data in [5].
S. Watanabe et al. L-Lactaldehyde dehydrogenase
FEBS Journal 275 (2008) 5139–5149 ª2008 The Authors Journal compilation ª2008 FEBS 5143