©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Molecular Cloning of the Isoquinoline 1-Oxidoreductase Genes from Pseudomonas diminuta 7, Structural Analysis of IorA and IorB, and Sequence Comparisons with Other Molybdenum-containing Hydroxylases (*)

Martin Lehmann , Barbara Tshisuaka , Susanne Fetzner , Franz Lingens (§)

From the (1)Institut für Mikrobiologie(250) , Universität Hohenheim, D-70593 Stuttgart, Federal Republic of Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 () 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.


INTRODUCTION

Isoquinoline 1-oxidoreductase (IOR)()from Pseudomonas diminuta 7 catalyzes the hydroxylation of isoquinoline to 1-oxo-1,2-dihydroisoquinoline with subsequent reduction of a suitable electron acceptor utilizing HO 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).

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 (/), 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.

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.


EXPERIMENTAL PROCEDURES

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).


RESULTS

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).

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-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.

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).

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.

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-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.

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%.


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).

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, 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).

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.


DISCUSSION

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 () 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.

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 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).

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 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)).

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.


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.


FOOTNOTES

*
This work was supported by the Ruetgerswerke AG, Castrop-Rauxel, Germany, the Fonds der Chemischen Industrie, and the Deutsche Forschungsgemeinschaft. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 49-711-459-2222; Fax: 49-711-459-2238.

The abbreviations used are: IOR, isoquinoline 1-oxidoreductase; PAGE, polyacrylamide gel electrophoresis; ORF, open reading frame.

J. Finsterbusch, R. Kappl, J. Hüttermann, M. Lehmann, B. Tshisuaka, S. Fetzner, and F. Lingens, unpublished results.


ACKNOWLEDGEMENTS

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.


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