From the
The iorA and iorB genes from the
isoquinoline-degrading bacterium Pseudomonas diminuta 7,
encoding the heterodimeric molybdo-iron-sulfur-protein isoquinoline
1-oxidoreductase, were cloned and sequenced. The deduced amino acid
sequences IorA and IorB showed homologies (i) to the small (
Isoquinoline 1-oxidoreductase (IOR)
Two eucaryotic members of molybdenum-containing
hydroxylases are known now, namely, the aldehyde oxidase and the
xanthine dehydrogenase (Wootton et al., 1991). They consist of
two 150-kDa subunits, which contain three domains of 20, 40, and 85 kDa
that were suggested to bear the [2Fe-2S] iron-sulfur
clusters, FAD, and the pterin molybdenum cofactor, respectively (Amaya et al., 1990; Wootton et al., 1991). Contrary to the
eucaryotic enzymes, the procaryotic molybdenum-containing hydroxylases
are structurally more diverse. There are complex hydroxylases
(
The
sequences of some molybdenum-containing hydroxylases have been
published recently, namely nicotine dehydrogenase from Arthrobacter
nicotinovorans (Grether-Beck et al., 1994), CO
dehydrogenase from Pseudomonas thermocarboxydovorans (Pearson et al., 1994), aldehyde oxidoreductase from D. gigas (Thoenes et al., 1994), part of the aldehyde
dehydrogenase genes from A. polyoxogenes (Tamaki et
al., 1989), and several eucaryotic xanthine dehydrogenases (Keith et al., 1987; Lee et al., 1987; Houde et
al., 1989; Riley, 1989; Amaya et al., 1990; Terao et
al., 1992; Xu et al., 1994).
In this paper we report
the cloning, sequence, and comparative sequence analysis of the iorA and iorB genes, encoding the isoquinoline
1-oxidoreductase of P. diminuta 7. Putative cofactor binding
sites were detected in the deduced amino acid sequence of iorA and iorB. In IorB, conserved domains were identified that
contained several highly conserved amino acid residues. These conserved
domains differ in their sequential arrangement among the compared
molybdenum-containing hydroxylases. Based on alignments of these
domains, the phylogenetic relationship of procaryotic and eucaryotic
molybdenum-containing hydroxylases is discussed.
iorA (456
base pairs) codes for a polypeptide (IorA) of 152 amino acids (Fig. 2) with a calculated isoelectric point of pH 7.84. The
predicted molecular mass of 16,399 Da corresponded to the molecular
mass of the small subunit of IOR (16 kDa) as estimated by SDS-PAGE
(Lehmann et al., 1994). The putative ribosome binding sequence
5`-AAAGA-3` preceded the ATG translational start codon (Fig. 2).
The N-terminal amino acid residues of the small subunit of IOR as
determined by Edman degradation coincided with the deduced amino acid
sequence Met
The codon usage of iorA and iorB agreed with the codon usage of pseudomonads as tabulated by Wada et al. (1992).
Upstream from the iorA and iorB genes, a further ORF, designated ORF2, was identified (1203 base
pairs, 401 amino acid residues). Comparison of ORF2 with DNA and
protein data bases revealed homologies to a number of ubiquinone
oxidoreductases, for instance to NADH dehydrogenase I chain F from E. coli (Weidner et al., 1993), which showed amino
acid identity to ORF2 of about 30%.
The C-terminal part of
an ORF (504 base pairs), designated ORF1, was detected upstream from
ORF2 (Fig. 2). The deduced amino acid sequence showed similarity
to several regulatory (DNA-binding) proteins. Proteins that regulate
the arabinose utilization (araC) in E. coli (Wallace et al., 1980) and Salmonella typhimurium (Clarke et al., 1992) were among the top matches. However,
determination whether ORF1 and ORF2 are involved in the regulation and
function of IOR will have to await further studies.
The G + C content of the sequenced DNA fragment was 64.48%. iorA and iorB showed G + C contents of 64.3% and
65.9%, respectively. For the genome of P. diminuta, a G
+ C content of 66.3-67.3% was reported by
Palleroni(1984).
The N-terminal 20 amino acid residues
of the small subunit, the N-terminal 28 amino acid residues of
fragment b, and the N-terminal 32 amino acid residues
of fragment d corresponded to Met
As
shown in Fig. 4A, IorA was homologous to the small
subunit of CO dehydrogenase from P. thermocarboxydovorans C2
(CutC) (Pearson et al., 1994), to the small subunit of
nicotine dehydrogenase from A. nicotinovorans (NdhB)
(Grether-Beck et al., 1994), to the N-terminal region
of the homodimeric aldehyde oxidoreductase from D. gigas (MOP)
(Thoenes et al., 1994), and to the N-terminal domain
of all eucaryotic xanthine dehydrogenases sequenced so far. These
sequences exhibited amino acid identity to IorA of 30-40%.
12 base
pairs downstream from the stop codon of the esterase V gene of Pseudomonas spec. KWI-56, an ORF (Est5) of unknown function
started (Shimada et al., 1993). The amino acid sequence of
Est5 was homologous to amino acid residues 1-88 of IorA and
contained all the conserved residues of this region detected by
aligning the sequences of published molybdenum-containing hydroxylases (Fig. 4A).
In the sequences aligned in Fig. 4A, 8 out of 29 conserved amino acids were cysteine
residues. The region containing Cys
The amino acid sequence deduced from iorB was homologous to the large subunit of CO dehydrogenase from P. thermocarboxydovorans C2 (CutA) (Pearson et al.,
1994), to the large subunit of nicotine dehydrogenase from A.
nicotinovorans (NdhC) (Grether-Beck et al., 1994), to the C-terminal region of aldehyde oxidoreductase from D. gigas (MOP) (Thoenes et al., 1994), and to the C-terminal domain of all eucaryotic xanthine dehydrogenases (Fig. 4C). The alignment revealed highest homology (30%
amino acid identity) of IorB with Adh from A. polyoxogenes (Tamaki et al., 1989). The N-terminal region of
Adh contains a signal peptide of 44 amino acids, showing features of
leader peptides important for transmembrane protein transport (Tamaki et al., 1989). The N-terminal stretch of IorB
strikingly resembles this leader region of Adh (Fig. 4D), which indicates that IOR is also a
membrane-associated protein.
Sequence similarities of IorB (Fig. 4C) were clustered in five regions, designated
domains A, B1, B2, C, and D (Fig. 3), which
were separated by regions without any homologies. Domains B1
and B2 were localized on fragment b, obtained after
trypsin treatment of IOR. Trypsin-generated fragment a
contained domain A. The C-terminal region of IorB,
which could not be assigned to a trypsin-generated fragment, harbored
domain C. Domains D, B1, and B2
were localized on fragment d, obtained by treatment of IOR
with trypsin (Fig. 3). As also shown in Fig. 3, the
arrangement of these distinct domains differs in IorB (A-D-B1-B2-C) and rat
xanthine dehydrogenase (D-B1-B2-A-C). Whereas Adh
showed the same sequential arrangement of domains as IorB, in xanthine
dehydrogenases, NdhC, CutA, and MOP, the region homologous to domain A was localized in the middle of the sequence. Since the
position of domain A (amino acid residues 39-113 of
IorB) differed in the aligned sequences, only the
``D-B1-B2'' unit, corresponding to
amino acid residues 230-603 of IorB, was conserved throughout.
The structural genes iorA and iorB from P. diminuta 7 were cloned and sequenced. We concluded from the
following observations that iorA and iorB code for
IOR: (i) the predicted molecular masses of IorA and IorB corresponded
to the molecular masses of the small and large subunit of IOR as
estimated by SDS-PAGE (Lehmann et al., 1994); (ii) the N-terminal amino acid sequences of the small subunit of IOR
and the N-terminal amino acid sequences of trypsin-generated
fragments of the large subunit of IOR, as determined by Edman
degradation, were identical with corresponding stretches of the
predicted amino acid sequences of the iorA and iorB genes; and (iii) the amino acid sequences deduced from iorA and iorB showed overall homologies to published sequences
of procaryotic and eucaryotic molybdenum-containing hydroxylases that
possess a pterin molybdenum cofactor with a monooxo-monosulfido-type
molybdenum center.
Comparison of IorB with other
molybdenum-containing hydroxylases revealed closest similarity with
aldehyde dehydrogenase from the acetic acid bacterium A.
polyoxogenes, which previously had been assumed to be a monomeric
pyrroloquinoline quinone-containing enzyme (Fukaya et al.,
1989a, 1989b). However, SDS-PAGE of purified aldehyde dehydrogenase
revealed a 75- and a 19-kDa peptide, and both the absorption spectrum
of the purified enzyme and the fluorescence spectrum of the large
peptide Adh (Fukaya et al., 1989a) were indicative for a
molybdenum-containing hydroxylase.
The adh gene
investigated by Tamaki et al.(1989) probably encodes a large
subunit, and the incompletely determined ORF upstream from adh, tentatively designated AdhA, which showed amino acid
identity of about 50% with the C-terminal part of IorA (see Fig. 4A), possibly codes for a small subunit,
respectively, of a putatively heterodimeric molybdenum-containing
aldehyde dehydrogenase. Thus, aldehyde dehydrogenase from A.
polyoxogenes (Fukaya et al., 1989a), quinaldic acid
4-oxidoreductase from S. marcescens 2CC-1 (Fetzner and
Lingens, 1993), and IOR from P. diminuta 7 are proposed to be
structurally related, since all of them are probably composed of a
small (16-18-kDa) and a large (75-80-kDa) subunit and since
they probably all contain [2Fe-2S] centers and a pterin
molybdenum cofactor but lack FAD.
The aldehyde dehydrogenase from A. polyoxogenes is a membrane-bound enzyme. The N-terminal region of IorB (amino acids 1-40) exhibited
significant homology to the N-terminal region of Adh (amino
acids 1-44), which contains a signal peptide (Fig. 4D) (Tamaki et al., 1989). We tentatively
suggest that the N-terminal region of IorB also contains a
signal peptide that is processed in mature IOR, since this region
showed essential features of typical signal peptides, such as the
presence of positive charges in the hydrophilic extreme N
terminus of IorB, followed by a hydrophobic region, which is able to
form the hydrophobic core of the putative signal peptide. Furthermore,
there was a typical arrangement of small neutral amino acid residues
around the potential cleavage site of processing. However, the proposed
signal peptide of IorB, comprising 40 amino acid residues, was longer
than other known signal peptides (15-25 amino acids) (von Heijne,
1984; Pugsley and Schwartz, 1985). Processing of IorB also would
explain the difference between the predicted molecular mass of IorB
(84,429 Da) and the molecular mass of the large subunit of IOR, as
estimated by SDS-PAGE (80 kDa). Association of IOR with the cell
membrane seems plausible, since possible electron acceptors of IOR such
as quinones or cytochromes (Lehmann et al., 1994) generally
are located in the membrane. Association with the cell membrane is
probably a common feature of procaryotic molybdenum-containing
hydroxylases. For instance, nicotine dehydrogenase from A.
nicotinovorans (Grether-Beck et al., 1994) and CO
dehydrogenase from Pseudomonas carboxydovorans (Rohde et
al., 1984) were reported to be membrane-associated proteins.
Apart from the high similarity of IorA and IorB with the two Adh
peptides from A. polyoxogenes, there were significant
homologies of IorA and IorB to the small (
None of the regions of IOR
corresponded to the middle domain of xanthine dehydrogenases.
Analogously, Thoenes et al.(1994) also did not detect a region
in aldehyde oxidoreductase from D. gigas that was homologous
to the middle domain of eucaryotic xanthine dehydrogenases. The
homodimeric enzyme from D. gigas, which contains two distinct
[2Fe-2S] clusters and a pterin molybdenum cofactor
(Moura et al., 1976; Romao et al., 1993), is composed
of two segments, an N-terminal stretch of amino acids
comprising residues 1-156, and a C-terminal region,
starting at amino acid 168 of the D. gigas enzyme, which were
homologous to IorA and IorB, respectively.
In eucaryotic xanthine
dehydrogenases, FAD was localized on the middle domain (Amaya et
al., 1990, Wootton et al., 1991). By analogy, it may be
assumed that the
Based on EPR studies,
eucaryotic xanthine dehydrogenases (Bray, 1975), aldehyde
oxidoreductase from D. gigas (Bray et al., 1991),
quinoline 2-oxidoreductase from P. putida 86 (Tshisuaka et
al., 1993), and IOR from P. diminuta 7
Since exactly eight cysteine
residues, which were conserved in all aligned sequences of different
molybdenum-containing hydroxylases, are present in IorA, cysteine
residues 97, 100, 132, and 134 of IorA are suggested to ligate the Fe/S
II center. Alignments of aldehyde oxidoreductase from D. gigas (Thoenes et al., 1994) and CO dehydrogenase from P.
thermocarboxydovorans (Pearson et al., 1994) with
eucaryotic xanthine dehydrogenases revealed homologous cysteine
residues, proposed to ligate the Fe/S II cluster. However, the proposed
Fe/S II binding region of IorA had no similarity to any other sequence
of an iron-sulfur protein deposited in DNA or protein data bases.
The C-terminal domain of eucaryotic xanthine dehydrogenases
(Amaya et al., 1990; Wootton et al., 1991), the C-terminal region of the aldehyde oxidoreductase of D.
gigas (Thoenes et al., 1994), and the large subunit of
procaryotic molybdenum-containing hydroxylases (Pearson et
al., 1994; Grether-Beck et al., 1994) have been proposed
to contain the molybdenum cofactor. Alignment of these domains or
segments with IorB revealed five conserved domains, designated A, B1, B2, C, and D ( Fig. 3and 4). The B1 domain contained the highly
conserved motif GG(G/T)FG(G/Y/Q/N/R)(K/R) (Fig. 4C). Amaya et al.(1990) drew attention to
the homologous motif GGGFGG, comprising amino acid residues
783-788 of rat xanthine dehydrogenase, which resembles the
consensus sequence GXGXXG of dinucleotide binding
proteins, and which was suggested to be involved in binding of a
dinucleotide cofactor or, possibly, the pterin molybdenum cofactor
(Amaya et al., 1990). It is important to note that all
eucaryotic xanthine dehydrogenases contain molybdopterin and not one of
the molybdopterin dinucleotides (Rajagopalan and Johnson, 1992).
Pearson et al.(1994), studying CO dehydrogenase from P.
thermocarboxydovorans, and Grether-Beck et al.(1994),
investigating nicotine dehydrogenase from A. nicotinovorans,
also associated this motif with binding of the molybdenum cofactor;
however, these enzymes contain a dinucleotide form of the molybdenum
cofactor (Grether-Beck et al., 1994; Pearson et al.,
1994). The multiple alignment shown in Fig. 4C reveals
that just the three glycine residues typical for the
GXGXXG consensus sequence of dinucleotide-binding
proteins were not conserved in the corresponding motif of procaryotic
molybdenum-containing hydroxylases. Furthermore, Hughes et
al.(1992), investigating strains of Drosophila
melanogaster, which carried a point mutation in the rosy gene, showed that a mutation leading to a substitution of the
first glycine residue of the GXGXXG motif in xanthine
dehydrogenase of D. melanogaster only caused a subtle
structural change of the enzyme but did not affect binding of the
molybdenum cofactor. Thus, the function and relevance of this highly
conserved sequence, GG(G/T)FG(G/Y/Q/N/R)(K/R), still
remains obscure.
Hughes et al.(1992) proposed a larger
region, ranging from amino acid residue 724 to 821 of rat liver
xanthine dehydrogenase, enclosing the motif discussed above, to be
involved in binding of the molybdenum cofactor. However, the alignment
of 12 sequences of molybdenum-containing hydroxylases, shown in Fig. 4C, revealed that in comparison with domains B2, D, and B1, which includes the region
emphasized by Hughes et al.(1992), the region, designated
domain C, contained the highest number of conserved amino acid
residues.
Domain C possessed a G-rich region (Fig. 4C, general amino acids 27-63), which
appeared similar to the G-rich regions found in nucleotide binding
sites (Wierenga et al., 1985). Surprisingly, the pattern of
conserved or conservatively changed amino acid residues of domain C, especially the G-rich region and the four amino acid
residues 737-751 of IorB, also showed similarity to a region of
IorA containing the Fe/S I center. However, alignment of these G-rich
regions did not reveal any remarkable differences between enzymes
reported to possess molybdopterin and those with a dinucleotide form of
the pterin molybdenum cofactor. Thus, it is still unclear whether
domain C, which shows a number of highly conserved amino acid
residues, is indeed involved in binding of the pterin molybdenum
cofactor.
Based on the comparisons of published sequences, it was
not possible to define distinct amino acid residues that might
contribute to ligation of the molybdenum atom. Thiolate groups from
cysteine residues were discussed as additional ligands of the
molybdenum center (Wootton et al., 1991), but there were no
cysteine residues conserved in the sequences compared with IorB. It may
be speculated that conserved threonine, tyrosine, aspartate, glutamate,
or glutamine residues (Fig. 4C) might contribute O- or N-ligands to the molybdenum center (Hille,
1994). However, further discussion of a possible binding site for the
pterin molybdenum cofactor and of possible additional ligands of the
molybdenum center will have to await further studies, especially x-ray
structural analyses of molybdenum-containing hydroxylases.
In order
to investigate the relationship of various procaryotic and eucaryotic
molybdenum-containing hydroxylases, a phylogenetic tree of the amino
acid region between amino acid residues 230 and 603 of IorB, which
comprises the domains D-B1-B2 (see Fig. 3), was established. As shown in Fig. 5, IorB from P. diminuta and Adh from A. polyoxogenes apparently
form a distinct subgroup. The relationship of Adh and IorB is also
illustrated by their identical sequential arrangement of the conserved
domains (A-D-B1-B2-C),
whereas in all other enzymes, the relative position of domain A differs from IorB and Adh (see Fig. 3). Nicotine
dehydrogenase from A. nicotinovorans (Grether-Beck et
al., 1994) and CO dehydrogenase from P. thermocarboxydovorans (Pearson et al., 1994) appeared to belong to another
distinct subgroup. In aldehyde oxidoreductase from the anaerobic,
sulfate-reducing bacterium D. gigas, however, the amino acid
sequence containing the D-B1-B2 unit was
more closely related to the corresponding region of eucaryotic xanthine
dehydrogenases than to those of other procaryotic molybdenum-containing
hydroxylases. The relative length of the branches symbolizing
eucaryotic xanthine dehydrogenases (Fig. 5) reflects the
phylogenetic distance of the corresponding organisms that served as
sources of the respective xanthine dehydrogenases.
We are grateful to Baerbel Haak and Frank Groß
for introduction to the methods of genetic engineering and to Prof.
Karl-Heinz van Pée (Dresden, Germany), who was generous with his
time for expert advice and discussion. We also thank Prof. Jung and S.
Stefanovic (Tübingen, Germany) for performing N-terminal
amino acid sequencing and Prof. Geldermann (Hohenheim, Germany) for
synthesis of oligonucleotides.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
) and
large (
) subunits of complex molybdenum-containing hydroxylases
(
/
)
possessing a pterin molybdenum cofactor with a monooxo-monosulfido-type
molybdenum center, (ii) to the N- and C-terminal regions of aldehyde
oxidoreductase from Desulfovibrio gigas, and (iii) to the N-
and C-terminal domains of eucaryotic xanthine dehydrogenases,
respectively. The closest similarity to IorB was shown by aldehyde
dehydrogenase (Adh) from the acetic acid bacterium Acetobacter
polyoxogenes. Five conserved domains of IorB were identified by
multiple sequence alignments. Whereas IorB and Adh showed an identical
sequential arrangement of these conserved domains, in all other
molybdenum-containing hydroxylases the relative position of
``domain A'' differed. IorA contained eight conserved
cysteine residues. The amino acid pattern harboring the four cysteine
residues proposed to ligate the Fe/S I cluster was homologous to the
consensus binding site of bacterial and chloroplast-type
[2Fe-2S] ferredoxins, whereas the pattern including the
four cysteines assumed to ligate the Fe/S II center showed no
similarities to any described [2Fe-2S] binding motif.
The N-terminal region of IorB comprised a putative signal peptide
similar to typical leader peptides, indicating that isoquinoline
1-oxidoreductase is associated with the cell membrane.
(
)from Pseudomonas diminuta 7 catalyzes the hydroxylation of
isoquinoline to 1-oxo-1,2-dihydroisoquinoline with subsequent reduction
of a suitable electron acceptor utilizing H
O as the source
of the oxygen atom incorporated into substrate. IOR is a heterodimeric
molybdo-iron-sulfur protein, composed of one 16- and one 80-kDa
subunit. Two distinct [2Fe-2S] iron-sulfur clusters,
designated Fe/S I and Fe/S II, were identified by EPR
spectroscopy.
(
)The pterin molybdenum cofactor of
IOR includes a molybdopterin cytosine dinucleotide and a
monooxo-monosulfido-type molybdenum center. The structural and
catalytic properties of IOR were described recently (Lehmann et
al., 1994).
/
),
which contain a pterin molybdenum cofactor, iron-sulfur clusters, and
FAD (Meyer, 1982; Freudenberg et al., 1988; Bauder et
al., 1990; Nagel and Andreesen, 1990; Peschke and Lingens, 1991;
Bauer and Lingens, 1992; de Beyer and Lingens, 1993; Sauter et
al., 1993; Kretzer et al.., 1993). These procaryotic
enzymes and the eucaryotic molybdenum-containing hydroxylases appear to
be structurally related. Besides these complex enzymes, additionally,
less complex procaryotic molybdenum-containing hydroxylases are
described that all lack the flavin. IOR from P. diminuta 7,
the heterodimeric (
) quinaldic acid 4-oxidoreductase from Serratia marcescens 2CC-1 (Fetzner and Lingens, 1993), the
homodimeric (
) aldehyde oxidoreductase from Desulfovibrio gigas (Romao et al., 1993), and
possibly the aldehyde dehydrogenase from Acetobacter polyoxogenes (Fukaya et al., 1989a) belong to this subgroup.
Materials
[-
S]dATP
was obtained from Amersham Corp. Restriction endonucleases, T4 DNA
ligase, T7 sequencing kit, and Deaza T7 sequencing mixes were from
Pharmacia Biotech Inc. DIG oligonucleotide 3`-end labeling kit, DIG
luminescent detection kit, and calf alkaline phosphatase were purchased
from Boehringer Mannheim.
Bacterial Strains, Plasmids, and Growth
Conditions
P. diminuta 7 was isolated from soil by
selective enrichment on isoquinoline as sole source of carbon and
energy (Röger et al., 1990). Growth conditions to obtain
cells for protein purification were described previously (Lehmann et al., 1994). For the preparation of DNA, strain 7 was grown
in LB medium (Sambrook et al., 1989) at 30 °C. Escherichia coli TG2 (Benen et al., 1989), which was
routinely grown in LB medium, and E. coli MV1190 (Vieira and
Messing, 1987) were used as standard host strains for cloning
experiments. Recombinant E. coli MV1190 clones were grown at
37 °C in 2 TY or H medium (Miller, 1972). Ampicillin was
added to the medium at a final concentration of 100 µg/ml. The
vectors used were pUC19, M13 mp18, and M13 mp19 (Messing et
al., 1977; Vieira and Messing, 1982; Norrander et al.,
1983; Yanisch-Perron et al., 1985).
DNA Techniques
Genomic DNA of P. diminuta 7 was extracted according to Davis et al.(1980).
Recombinant plasmid DNA from E. coli clones was isolated by
the alkaline lysis method (Sambrook et al., 1989). Agarose gel
electrophoresis, DNA restriction, treatment with alkaline phosphatase,
and DNA ligation were done by standard procedures (Sambrook et
al., 1989). DNA fragments were isolated from agarose gels
according to Tautz and Renz(1983). Transformation of E. coli with plasmid DNA was performed by the CaCl method
(Mandel and Higa, 1970). DNA was transferred from agarose gels to nylon
membranes (Hybond-N, Amersham Corp.) as described by the manufacturer. Preparation and Screening of an Enriched Gene Library of P. diminuta 7
DNA-P. diminuta 7 DNA was totally digested with SacI.
DNA fragments of 7000-10,000 base pairs were isolated from an
agarose gel and ligated in SacI-digested and dephosphorylated
pUC19. E. coli TG2 was transformed with the ligated DNA.
Recombinant clones were detected by colony hybridization (Grunstein and
Hogness, 1975), using the mixed oligonucleotide ATG AT(C/T) GA(A/G) TTC
AT(C/T) CT(G/C) AA(C/T) GGC CAG CC, which was labeled with the DIG
oligonucleotide 3`-end labeling kit as described by the supplier, as a
probe. The oligonucleotide had been deduced from the N-terminal amino acid residues
Met
-Pro
of the small subunit of IOR.
Hybridizations were carried out at 55 °C in 2
SSC (Sambrook et al., 1989) for 17 h. The nylon membranes were stringently
washed twice for 10 min at 58 °C in 1
SSC containing 0.1%
SDS and once for 5 min at 42 °C in 0.1
SSC containing 0.1%
SDS. Immunological detection was performed with the DIG luminescent
detection kit.
DNA Sequencing and Sequence Analysis
DNA
sequencing was done by the dideoxy method (Sanger et al.,
1977). Specific fragments, comprising the iorA and iorB genes and flanking regions, were subcloned in M13 mp18 and M13
mp19. Transfection of E. coli with M13 phage DNA, preparation
of single stranded DNA, and dideoxy sequencing reactions were performed
according to the [-
S]dATP sequencing kit
instruction manual (Pharmacia). All stretches of DNA were sequenced in
both directions. Sequence ladders were resolved on denaturing gels
containing 6% (w/v) polyacrylamide. The strategy of DNA sequencing is
outlined in Fig. 1. Computer analyses of the DNA sequences were
performed with the GENMON program (Gesellschaft für
Biotechnologische Forschung, Braunschweig, Germany) and the HUSAR 3.0
program package (EMBL, Heidelberg, Germany), which includes the GCG
software version 7.1 of the University of Wisconsin (Devereux et
al., 1984). PILEUP and CLUSTALV (Higgins and Sharp, 1988) were
used to calculate multiple alignments of IorA and IorB, respectively,
whereas the alignments of IorB with Adh were performed using GAP.
Calculation of cladograms was done by TREE.
Figure 1:
Restriction map
and sequencing strategy for the DNA fragment containing iorA, iorB, and two further open reading frames. Only the
restriction sites used for sequencing and subcloning are given. Arrows indicate the length and sequencing direction of
fragments subcloned in M13 mp18 and M13 mp19. The boxes indicate the position and the direction of transcription of the
open reading frames.
Trypsin Digestion of IOR
IOR was purified as
described previously (Lehmann et al., 1994). 1 mg of purified
enzyme was incubated at 37 °C for 1 h with 0.1 mg of trypsin
(Sigma) in 50 mM Tris/HCl, pH 8.0. An aliquot of the digested
enzyme was applied to a Superdex 200 prep grade HiLoad 16/60 column
(Pharmacia) equilibrated in 100 mM Tris/HCl buffer, pH 8.5.
Protein Sequence Analysis
IOR (as isolated) and
trypsin-digested IOR were separated by SDS-PAGE (Schägger and von
Jagow, 1987) and electroblotted onto a polyvinylidene difluoride
membrane as recommended by Pharmacia bulletin S.D. RE-072. Amino acid
sequences of N-terminal and internal peptide fragments were determined
by automated Edman degradation (sequencer model 477A, equipped with
analyzer model 120A, Applied Biosystems, Weiterstadt, Germany).
Enzyme Assay
The IOR activity was determined
spectrophotometrically, following the formation of
2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium
chloride formazan at 503 nm as described previously (Lehmann et
al., 1994).
Nucleotide Sequence Accession Number
The DNA
sequence presented in this report, encoding open reading frame (ORF) 1,
ORF2, and iorA and iorB, has been deposited in the
EMBL Nucleotide Sequence Library, Heidelberg, Germany, under accession
number Z48918 (PDIORAB).
Cloning and Sequencing of IOR and Flanking
Regions
An enriched gene library of genomic P. diminuta 7 DNA in pUC19 was screened for iorA and iorB using a mixed oligonucleotide that corresponded to the first 10 N-terminal amino acid residues of the small subunit of IOR as
a probe. An 8.5-kilobase pair DNA fragment, designated pML8.5, was
isolated. A restriction map of a part of pML8.5 is shown in Fig. 1. This figure also outlines the strategy used to determine
both strands of the iorA and iorB genes and their
flanking regions. Three ORFs, designated ORF2, iorA, and iorB, and part of another ORF (ORF1) were detected on pML8.5 (Fig. 1) by the computer programs FRAMES, TESTCODE, and
CODONPREFERENCE, the last being applied with the codon usage table of
the pseudomonads (Wada et al., 1992).
-Ala
.
Figure 2:
Nucleotide sequence of iorA and iorB and flanking regions. The deduced amino acid sequences
are shown below the DNA sequence. Underlined amino
acid sequences are identical to those determined by Edman degradation.
Potential ribosome binding sites (marked as SD, boldface), start codons (boldface), and stop codons (threeasterisks) are indicated.
iorB (Fig. 2) comprised 2343 base pairs of pML8.5. The gene
overlapped four base pairs with iorA (Fig. 2). The
primary structure of the iorB polypeptide consisted of 781
amino acid residues, corresponding to 84,429 Da, which largely matches
the molecular mass of the large subunit of IOR as estimated by SDS-PAGE
(80 kDa) (Lehmann et al., 1994). The sequence 5`-AAGGAGG-3`
preceding the start codon ATG of iorB probably served as the
ribosome binding site (Fig. 2). IorB had a predicted isoelectric
point of pH 8.55.
Trypsin Digestion of IOR
In order to prove the
assignment of iorB to the large subunit of IOR, purified
enzyme (1 mg) was treated with trypsin, and generated fragments were
subjected to N-terminal amino acid sequence analysis. After
trypsin treatment, IOR lost 30% of its specific activity, but, as gel
filtration revealed, no peptide fragments dissociated from the enzyme.
In SDS-PAGE, however, trypsin-treated IOR separated into five major
protein bands: fragments a (19.6 kDa), b (33.6 kDa), d (53 kDa), and e (67 kDa), which were derived from
the large subunit, and the small subunit, which apparently was not
attacked by trypsin.
-Ala
of IorA, to Asn
-Phe
of IorB,
and to Asn
-Leu
of IorB, respectively (Fig. 2). These findings proved the assignment of iorA and iorB to the small and large subunit of IOR,
respectively. The N termini of the large subunit and of
fragments b and d were not accessible to Edman
degradation. Based on the N-terminal amino acid sequences and
based on the molecular masses of the trypsin-generated fragments, a
scheme for trypsin digestion of IorB is outlined in Fig. 3.
Time-limited trypsin treatment revealed that the site most accessible
to trypsin cleavage was the peptide bond between amino acids
Arg
-Val
, yielding fragment e and a C-terminal fragment that was not detected by SDS-PAGE. The
second cleavage site occurred between Arg
and
Asp
, resulting in fragments a and d.
Sites 3 and 4 shortened fragment d to fragments c and b, respectively (Fig. 3). In fragments a and d, no further cleavage sites were accessible to trypsin
digestion.
Figure 3:
Peptide fragments obtained after trypsin
treatment of IOR, and arrangement of conserved domains in IorB and in
rat liver xanthine dehydrogenase (Amaya et al., 1990). Largeboxes (upperpart) indicate
the peptide fragments of IOR generated by successive trypsin attack,
indicated by numbersinparentheses. Numbers
of amino acid residues are boldface. Smallboxes, labeled A, B1, B2, C, and D, indicate the length and position of
conserved domains. XDH_rat, rat xanthine
dehydrogenase.
Multiple Alignments of Molybdenum-containing
Hydroxylases
Comparison of the deduced amino acid sequence of
IorA and IorB with published sequences of other molybdoenzymes
containing the pterin molybdenum cofactor revealed similarity only to
enzymes possessing a monooxo-monosulfido-type molybdenum center.
Figure 4:
Alignment of IorA (panel A) and
IorB (panel C) with corresponding regions of other
molybdenum-containing hydroxylases. Alignment of IorA and IorB was
performed using the PILEUP and CLUSTALV programs, respectively. Asterisks indicate amino acid residues that are conserved in
all the aligned sequences; dots indicate residues that are
identical or similar; and residues set in boldface are
identical or similar in all but one of the aligned sequences. Conserved
cysteines are set in lowercase. Adh, aldehyde
dehydrogenase from A. polyoxogenes (Tamaki et al.,
1989); Cut, CO dehydrogenase from P. thermocarboxydovorans (Pearson et al., 1994); Ndh, nicotine
dehydrogenase from A. nicotinovorans (Grether-Beck et
al., 1994); MOP, aldehyde oxidoreductase from D.
gigas (Thoenes et al., 1994); XDH_man, human
xanthine dehydrogenase (Xu et al., 1994)); XDH_rat, rat xanthine dehydrogenase (Amaya et
al., 1990); XDH_mouse, mouse xanthine dehydrogenase
(Terao et al., 1992); XDH_chic, chicken xanthine dehydrogenase
(EMBL accession: GGXDHY); XDH_drome, D.
melanogaster xanthine dehydrogenase (Keith et al., 1987;
Lee et al., 1987); XDH_drops, Drosophila
pseudoobscura xanthine dehydrogenase (Riley, 1989); XDH_calvi, Calliphora vicina xanthine dehydrogenase
(Houde et al., 1989); Est5, ORF of unknown function, located
downstream from the esterase V gene from Pseudomonas spec.
KWI-56 (Shimada et al., 1993). Panel B, comparison of
the putative [2Fe-2S] binding site Fe/S I of IorA with
[2Fe-2S] binding regions of bacterial ferredoxin
proteins. Identical amino acid residues are indicated using the
one-letter code of the amino acid, and similar amino acids are marked
(+). Panel D, homology of the signal peptide of Adh with
the N-terminal region of IorB. Identical amino acid residues
are indicated by verticallines. Conservative amino
acid replacements are marked by colons and dots.
Upstream from the adh gene from A. polyoxogenes,
proposed to encode an aldehyde dehydrogenase (see below), Tamaki et
al.(1989) had sequenced part of another potential ORF
(``AdhA''). The predicted amino acid sequence of this
incomplete ORF showed about 50% amino acid identity to a stretch of
IorA, starting at amino acid 78 (Fig. 4A).
, Cys
,
Cys
, and Cys
of IorA was homologous to the
[2Fe-2S] binding site of bacterial and chloroplast-type
ferredoxins (Wootton et al., 1991; Hughes et al.,
1992). However, the second region harboring the other four conserved
cysteine residues (amino acids 97, 100, 132, and 134 of IorA) exhibited
no similarity to any other reported Fe/S consensus sequence. These
cysteine residues probably ligate the second type of
[2Fe-2S] center, designated Fe/S II, which was detected
by EPR for IOR
and for other molybdenum-containing
hydroxylases (Bray, 1975, Bray et al., 1991, Tshisuaka et
al., 1993).
) and large (
)
subunits of molybdenum-containing hydroxylases possessing
or
structure, to the N- and C-terminal regions of
the homodimeric aldehyde oxidoreductase from D. gigas (Thoenes et al., 1994), and to the N- and C-terminal
domains of the eucaryotic xanthine dehydrogenases, respectively (Fig. 4). For instance, the N-terminal amino acids
1-165 and the C-terminal domain (starting at amino acid
residue 552) of rat liver xanthine dehydrogenase were homologous to
IorA and IorB, respectively. Boundaries for these three domains, as
defined in rat liver xanthine dehydrogenases by trypsin cleavage (Amaya et al., 1990), corresponded well with boundaries, as found by
the multiple alignments performed.
subunit of the structurally complex procaryotic
molybdenum-containing hydroxylases
(
/
)
bears the flavin. IOR from P. diminuta 7, quinaldic acid
4-oxidoreductase from S. marcescens 2CC-1, and aldehyde
oxidoreductase from D. gigas, which do not possess flavin,
also lack an intermediate-sized subunit or a corresponding middle
domain. This conclusion is confirmed by sequence analysis of two
complex procaryotic molybdenum-containing hydroxylases, namely the
nicotine dehydrogenase from A. nicotinovorans and the CO
dehydrogenase from P. thermocarboxydovorans, whose
subunits revealed striking similarity to the middle, FAD-bearing domain
of eucaryotic xanthine dehydrogenases (Grether-Beck et al.,
1994; Pearson et al., 1994).
were
shown to contain two distinct [2Fe-2S] clusters,
designated Fe/S I and Fe/S II. The N-terminal domains of
eucaryotic xanthine dehydrogenases, which were proposed to harbor these
[2Fe-2S] clusters Fe/S I and Fe/S II (Amaya et
al., 1990, Wootton et al., 1991), were homologous to
IorA. Four cysteine residues (amino acid residues 39, 44, 47, and 59 of
IorA) proposed to ligate the [2Fe-2S] cluster Fe/S I
were conserved in all published sequences of procaryotic and eucaryotic
molybdenum-containing hydroxylases (Fig. 4A). The
stretch of amino acids of IorA ligating these four cysteine residues
showed significant similarity to the motif of bacterial
[2Fe-2S] ferredoxins, to chloroplast-type ferredoxins
(Wootton et al., 1991), and to the [2Fe-2S]
binding region of bacterial succinate and fumarate dehydrogenases (Fig. 4B; see also Hughes et al.(1992) and
Grether-Beck et al.(1994)).
Figure 5:
Phylogenetic tree of the D-B1-B2 unit,
corresponding to amino acid residues 230-603 of IorB. The
phylogenetic tree was calculated by the program TREE of the GCG
software package. XDH_hxa, xanthine dehydrogenase
from Aspergillus nidulans (EMBL accession: ANHXA);
all other abbreviations are given in the legend of Fig. 4. The length
of the branches and the numbers indicate the relative
phylogenetic distance of the sequences.
DNA sequence
analysis of further procaryotic molybdenum-containing hydroxylases and,
especially, genetic studies such as site-directed mutagenesis of
putatively crucial amino acid residues would contribute to establish
the pattern of cofactor binding sites and to elucidate the structure
and function of the distinct conserved domains of the
molybdenum-containing hydroxylases.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.