This is a pre-copyedited, author-produced version of an article accepted for publication in Bioscience, Biotechnology, and Biochemistry following peer review.
The version of record "Eight genes are necessary and sufficient for biogenesis of quinohemoprotein amine dehydrogenase" is available online at: https://doi.org/10.1093/bbb/zbab117.
Eight Genes Are Necessary and Sufficient for Biogenesis of Quinohemoprotein Amine Dehydrogenase
Tadashi Nakai1,2,3,*, Katsuyuki Tanizawa3, Toshihide Okajima3
1Graduate School of Science and Technology, Hiroshima Institute of Technology, Hiroshima, Japan.
2Faculty of Life Sciences, Hiroshima Institute of Technology, Hiroshima, Japan.
3Institute of Scientific and Industrial Research, Osaka University, Osaka, Japan.
*Correspondence: Tadashi Nakai, t.nakai.wj@cc.it-hiroshima.ac.jp
Running Head
Heterologous expression of eight qhp genes
Abstract
Quinohemoprotein amine dehydrogenase (QHNDH) containing a peptidyl quinone cofactor, cysteine tryptophylquinone, is produced in the periplasm of Gram-negative bacteria through an intricate process of post-translational modification that requires at least eight genes including those encoding three nonidentical subunits and three modifying enzymes. Our heterologous expression study has revealed that the eight genes are necessary and sufficient for the QHNDH biogenesis.
Keywords
quinohemoprotein amine dehydrogenase, post-translational modification, biogenesis of cofactor, heterologous expression, amine assimilation
Quinohemoprotein amine dehydrogenase (QHNDH) is a widely distributed bacterial enzyme that catalyzes the oxidative deamination of primary amines, which are used as energy, carbon, and nitrogen sources (Adachi et al. 1998; Takagi et al. 1999; Takagi et al. 2001). The crystal structures of QHNDH (Datta et al. 2001; Satoh et al. 2002) revealed the common heterotrimeric subunit structure, consisting of three nonidentical subunits; the ~60-kDa α-subunit containing two c-type hemes, the ~37-kDa β-subunit, and the ~9-kDa γ-subunit containing a peptidyl quinone cofactor, cysteine tryptophylquinone (CTQ), and three intra-peptidyl thioether crosslinks formed between specific Cys and Asp/Glu residues. Thus, the small γ-subunit must undergo an intricate process of post-translational modification before maturation (Datta et al. 2001; Nakai et al. 2014).
Structural genes encoding QHNDH constitute an operon termed qhp, which together with several nearby genes forms a gene cluster (qhpABCDEFGR). We previously showed that these eight structural genes are essential for the amine-induced expression of the enzyme in the periplasm of a QHNDH-producing bacterium, Paracoccus denitrificans Pd1222 (Nakai et al. 2014). The qhp operon is distributed in more than 1,300 bacterial species, mostly belonging to Gram-negative bacteria (Oozeki et al. 2021). The qhpA, qhpB, and qhpC genes encode the α-, β-, and γ-subunits of QHNDH, respectively. The qhpD, qhpE, and qhpG genes encode the three enzymes involved in post-translational modification of QhpC (precursor to the mature γ-subunit); a radical S-adenosylmethionine (SAM) enzyme that catalyzes the sequential formation of three Cys-Asp/Glu thioether bonds in QhpC (Ono et al. 2006; Nakai et al. 2015), a subtilisin-like serine protease that cleaves the leader peptide at the N-terminal 28 residues of QhpC (Nakai et al. 2012), and an atypical single-component monooxygenase that catalyzes the dihydroxylation of a specific unmodified Trp residue in QhpC (Oozeki et al. 2021), respectively. The qhpF gene encodes an efflux ABC transporter that transfers the post-translationally modified QhpC into the periplasm (Nakai et al. 2014). The qhpR gene encodes an amine-inducible AraC-like transcriptional regulator that activates transcription of the qhp gene cluster (Nakai et al. 2014). In P. denitrificans Pd1222, the qhp genes are organized in an order of GADCBEFR on chromosome 1, and the qhpADCBEF and qhpG genes are transcribed individually under the common control of the qhp promoter (Nakai et al. 2014).
Periplasmic degradation of primary amines in Gram-negative bacteria starts with the oxidative deamination of their amino groups by amine dehydrogenase or amine oxidase, both forming ammonia and the corresponding aldehydes as the common reaction products (Hacisalihoglu et al. 1997). The degradation then continues with the cytoplasmic oxidation of the aldehydes by aldehyde dehydrogenase, followed by the transfer of coenzyme A (CoA) to the carboxylic acids. The resulting acyl-CoA derivatives are assimilated through the normal β-oxidation pathway. In P. denitrificans, the first step of amine oxidation is catalyzed by QHNDH that is inducibly formed by the amines added to the culture medium (Takagi et al. 1999).
As described above, biogenesis of QHNDH has an intricate mechanism, in which the eight genes of qhpABCDEFGR were shown to be essential (Nakai et al. 2014). However, it remains unclear whether they solely are sufficient or further genes are necessary for the QHNDH biogenesis. In this study, we expressed the eight qhp genes in a QHNDH-non-producing bacterium, Rhodobacter sphaeroides NRBC 12203, which lacks an amine assimilating ability without the qhp operon. The heterologous expression study reported herein reveals that the eight genes are sufficient for production of active QHNDH, conferring the amine assimilating ability on the host bacterium, though weakly. Additionally, the enzyme heterologously expressed in the periplasm of R. sphaeroides cells was shown to have a correctly post-translationally modified structure.
For heterologous expression of the eight qhp genes in R. sphaeroides, we constructed an expression plasmid, pRK-qhpGADCBEFR, and introduced the plasmid into R. sphaeroides by diparental mating using E. coli S17-1 as the donor cells. Further details of materials and methods are provided as the Supplementary material.
We first examined the growth of the transformant in the minimal medium containing n-butylamine (n-BA) as the sole carbon source (n-BA minimal medium), in which the qhp genes would be inducibly transcribed. As shown in Figure 1, R. sphaeroides cells without the plasmid did not grow at all in the n-BA minimal medium, in marked contrast to the good growth in the minimal medium containing glucose as a carbon source. By transformation with the eight qhp genes, R. sphaeroides cells began to grow in the n-BA minimal medium, although very slowly as compared to the rapid growth of the QHNDH-producing P. denitrificans Pd1222 cells. The QHNDH activities were also detected in the R. sphaeroides cells grown for >120 h (0.54 ± 0.06 U/g of wet cells, n = 2), which were comparable (~74%) to those of the QHNDH-producing P. denitrificans Pd1222 cells grown for 20–24 h (0.73 ± 0.06 U/g of wet cells, n = 3). These results show that transformation of R. sphaeroides with the eight qhp genes leads to the production of active QHNDH and thereby confers an ability to slowly assimilate n-BA as the sole carbon source on the bacterium.
Finally, to study the biogenesis of QHNDH in the R. sphaeroides cells transformed with the eight qhp genes, we cultured the cells of R. sphaeroides first in the minimal medium supplemented with glucose (to support the cell growth) followed by further incubation for 24–36 h in the glucose minimal medium added with 18 mM n-BA (to induce the QHNDH expression). We then purified the enzyme from the periplasmic fraction of the cells to conduct the structural analysis, which is the most direct way to confirm the full process of the QHNDH biogenesis, including transcription of all qhp genes, post-translational modification of the QhpC protein (precursor to the γ-subunit), periplasmic transportation of the three subunits, and heme insertion into the α-subunit together with the final CTQ formation in the γ-subunit (Nakai et al. 2014).
As expected, the enzymes purified from the periplasmic fractions of both of the P. denitrificans and R. sphaeroides cells had almost the same specific activities (3.8 U/mg protein for the enzyme purified from R. sphaeroides harboring pRK-qhpGADCBEFR and 3.9 U/mg protein for the enzyme purified from P. denitrificans) and identical heterotrimeric subunit compositions in the SDS-PAGE analysis (Figures 2a, 2b). In the mature γ-subunit, a quinone group derived from the CTQ cofactor was also detected (Figure 2c). Furthermore, UV-visible absorption spectra of the purified enzymes were identical with each other, both having the absorption maximum at 410 nm (Figures 3a, 3b), which is mainly due to two c-type hemes bound to the a-subunit, demonstrating the proper insertion of two heme groups into the a-subunit. Mass spectra of the isolated γ-subunits from both bacteria showed a common peak at m/z of about 8859 (Figures 3c, 3d), well corresponding to the calculated mass of the mature g-subunit (m/z, 8857.8) containing CTQ and three intrapeptidyl Cys-Asp/Glu crosslinks; no free Cys residues were contained as judged from the same mass peaks before and after the treatment with 2-iodoacetamide (IAA). These results unequivocally show that the recombinant QHNDH containing CTQ and three Cys-Asp/Glu crosslinks is correctly assembled even when it is heterologously expressed in R. sphaeroides.
Notwithstanding the periplasmic production of active QHNDH, R. sphaeroides cells carrying the eight qhp genes grow very slowly in the minimal medium containing n-BA as the sole carbon source, as described above. Two possible reasons for this slow growth may be considerable. One possibility is the metabolic inefficiency in the assimilation of the sole carbon (and also energy) source, n-BA, added to the culture medium, including the low ability of the downstream metabolism through n-butyraldehyde and n-butyric acid. Regardless of the expression of enzymatically active QHNDH, oxidation of n-BA in the periplasm requires an external electron acceptor that interacts with QHNDH, such as cytochrome c550 (serving in P. denitrificans; Takagi et al. 2001) and azurin (serving in Pseudomonas putida; Adachi et al. 1998), and transfers reducing energies needed for cell growth. The electron acceptor, if any, in the periplasm of R. sphaeroides cells may not function well with QHNDH, leading to the inefficient oxidation of n-BA that results in the less supply of carbon and energy sources. Furthermore, if the n-butyraldehyde produced from n-BA by the action of QHNDH does not serve as a good substrate for the next enzyme, aldehyde dehydrogenase, that may exist constitutively in R. sphaeroides cells (or it may be less abundant in the cells grown in the n-BA minimal medium), the accumulating n-butyraldehyde might strongly inhibit the cell growth, in marked contrast to the subsequent metabolite, n-butyric acid, that is assimilated very efficiently by R. sphaeroides cells (see Supplementary Figure S1).
Another possibility is the retarded post-translational modification of QhpC in the cells grown in the n-BA minimal medium, in particular the formation of three Cys-Asp/Glu crosslinks by the radical SAM enzyme QhpD containing multiple [Fe-S] clusters (Nakai et al. 2015) and the dihydroxylation of the CTQ-precursor Trp residue by the flavoprotein monooxygenase QhpG (Oozeki et al. 2021). For the QhpD and QhpG reactions to proceed, three and four electrons are required, respectively, but the physiological electron donors for the two reactions are unknown at present. Electron transfer proteins commonly existing in bacterial cells (e.g., ferredoxin, flavodoxin, and thioredoxin) that are encoded elsewhere in the genome may supply electrons for QhpD and/or QhpG. It is likely that such an electron transfer protein is little formed unless R. sphaeroides cells are grown in the medium containing a good carbon source, such as glucose. In addition, the systems of heme biosynthesis and those needed for insertion of hemes into the α-subunit of QHNDH as well as the transporters involved in the periplasmic translocation of the α- and β-subunits may be deficient in the R. sphaeroides cells cultured in the n-BA minimal medium, resulting in the slow biogenesis of QHNDH.
In conclusion, we have demonstrated that the eight genes of qhpABCDEFGR are the minimal gene set that is necessary for the QHNDH biogenesis. The eight qhp genes are also sufficient for the biogenesis, since other relating proteins, such as electron transfer proteins, are replaceable with those inherent in R. sphaeroides cells grown in the glucose minimal medium. A similar heterologous expression study using R. sphaeroides as the host cells was previously reported for identification of the genes required for the biogenesis of methylamine dehydrogenase containing a peptidyl quinone cofactor, tryptophan tryptophylquinone, that is produced by a complicated process of post-translational modification, being markedly different from that of the biogenesis of CTQ-containing QHNDH reported here (Graichen et al. 1999).
Acknowledgments
We thank the staff of the Comprehensive Analysis Center, Institute of Scientific and Industrial Research, Osaka University for technical assistance in mass spectrometric analysis.
Supplementary material
Supplementary material for this article can be found at the end of this file.
Author contribution
T.N., K.T., and T.O. participated in research design; T.N. conducted experiments; and T.N., K.T., and T.O. analyzed data and wrote the manuscript.
Funding
This research was supported by JSPS KAKENHI Grant Numbers JP 23570135 and JP 16K07691 to T.N., and JP 24658288 and JP 15K07391 to T.O., and by the Cooperative Research Program of “Network Joint Research Center for Materials and Devices”
Disclosure statement
The authors declare no competing interests.
Data Availability
The data underlying this article will be shared on reasonable request to the corresponding author.
References
Adachi O, Kubota T, Hacisalihoglu A et al. Characterization of quinohemoprotein amine dehydrogenase from Pseudomonas putida. Biosci Biotechnol Biochem 1998;62:469–78.
Datta S, Mori Y, Takagi K et al. Structure of a quinohemoprotein amine dehydrogenase with an uncommon redox cofactor and highly unusual crosslinking. Proc Natl Acad Sci USA 2001;98:14268–73.
Graichen ME, Jones LH, Sharma BV et al. Heterologous expression of correctly assembled methylamine dehydrogenase in Rhodobacter sphaeroides. J Bacteriol 1999;181:4216–22.
Hacisalihoglu A, Jongejan JA, Duine JA, Distribution of amine oxidases and amine dehydrogenases in bacteria grown on primary amines and characterization of the amine oxidase from Klebsiella oxytoca. Microbiology 1997;143:505–12.
Nakai T, Deguchi T, Frebort I et al. Identification of genes essential for the biogenesis of quinohemoprotein amine dehydrogenase. Biochemistry 2014;53:895–907.
Nakai T, Ito H, Kobayashi K et al. The radical S-adenosyl-L-methionine enzyme QhpD catalyzes sequential formation of intra-protein sulfur-to-methylene carbon thioether bonds, J Biol Chem 2015;290:11144–66.
Nakai T, Ono K, Kuroda S et al. An unusual subtilisin-like serine protease is essential for biogenesis of quinohemoprotein amine dehydrogenase. J Biol Chem 2012;287:6530–8.
Ono K, Okajima T, Tani M et al. Involvement of a putative [Fe-S]-cluster-binding protein in the biogenesis of quinohemoprotein amine dehydrogenase. J Biol Chem 2006;281:13672–84.
Oozeki T, Nakai T, Kozakai K et al. Functional and structural characterization of a flavoprotein monooxygenase essential for biogenesis of tryptophylquinone cofactor, Nat Commum 2021;12:933.
Satoh A, Kim JK, Miyahara I et al. Crystal structure of quinohemoprotein amine dehydrogenase from Pseudomonas putida. Identification of a novel quinone cofactor encaged by multiple thioether cross-bridges. J Biol Chem 2002;277:2830–4.
Takagi K, Torimura M, Kawaguchi K et al. Biochemical and electrochemical characterization of quinohemoprotein amine dehydrogenase from Paracoccus denitrificans. Biochemistry 1999;38:6935–42.
Takagi K, Yamamoto K, Kano K et al. New pathway of amine oxidation respiratory chain of Paracoccus denitrificans IFO 12442. Eur J Biochem 2001;268:470–6.
Figure legends
Figure 1
Growth of P. denitrificans and R. sphaeroides with or without QHNDH expression plasmid. Cells of P. denitrificans Pd1222 (■), R. sphaeroides (Rsp) (●), and Rsp transformed with pRK-qhpGADCBEFR (▲) were grown in the n-BA minimal medium. Cells of Rsp harboring no plasmid were also grown in the glucose minimal medium (○). Cell densities measured by OD600 were plotted against culture time (h).
Figure 2
SDS-PAGE analysis of purified QHNDH. The recombinant and native QHNDH were expressed in R. sphaeroides (Rsp) transformed with pRK-qhpGADCBEFR and P. denitrificans (Pd1222), respectively, and purified from the periplasmic fractions of each bacterium. The purified protein was subjected to SDS-PAGE followed by staining with Coomassie Blue for the α- and β-subunits (a) and Western blotting with an anti-γ-subunit antibody (Ono et al. 2006) for the γ-subunit that is hardly stained with Coomassie Blue (b). The same membrane was also used for detection of a redox active quinone group by quinone staining (c).
Figure 3
UV-visible absorption spectra of purified QHNDH and MALDI-TOF mass spectra of γ-subunit. UV-visible absorption spectra of the recombinant and native QHNDH purified from the cells of R. sphaeroides transformed with pRK-qhpGADCBEFR (a) and P. denitrificans (b), respectively, are shown after normalization to the same protein concentration (1 mg/mL). The γ-subunit isolated from the recombinant (c) and native (d) QHNDH by heat treatment was incubated with (+) or without (–) 50 mM 2-iodoacetamide (IAA) for 1 h, desalted with a C18 ZipTip pipette tip, and subjected to mass spectrometric analysis.
Supplementary material
Materials and methods
Materials, bacterial strains, and culture conditions
Escherichia coli strains DH5α and S17-1 were used for plasmid preparation and diparental mating, respectively. E. coli was grown aerobically at 37 °C in a Luria broth (LB) medium [1% (w/v) polypeptone, 0.5% (w/v) yeast extract, and 0.5% (w/v) NaCl]. Paracoccus denitrificans strain Pd1222 (Pd1222) and E. coli strain S17-1 were kindly provided by R. J. van Spanning (Vrije Universiteit, The Netherlands). Rhodobacter sphaeroides NBRC 12203 was obtained from the NITE Biological Resource Center (Chiba, Japan). R. sphaeroides and P. denitrificans were grown aerobically at 30 °C in an LB medium or a minimal mineral medium described below.
The minimal mineral medium contained (per liter of deionized water) 6 g of Na2HPO4, 3 g of KH2PO4, 0.5 g of NaCl, and 1 g of NH4Cl. The following components were sterilized separately and then added (per liter of final medium): 1 mL of 1 M MgSO4, 0.1 mL of 1 M CaCl2, 0.1 mL of vitamin solution (filter sterilized, see below) for R. sphaeroides, and 0.1 mL (for R. sphaeroides) or 1 mL (for P. denitrificans) of their respective trace solutions (see below). The vitamin solution contained (per 20 mL) 200 mg of nicotinic acid, 100 mg of thiamine-HCl, 2 mg of D-biotin, and 20 mg of p-amino benzoic acid. The trace solution for R. sphaeroides contained (per 100 mL) 1.765 g of EDTA, 10.95 g of ZnSO4·7H2O, 5.0 g of FeSO4·7H2O, 1.54 g of MnSO4·H2O, 0.392 g of CuSO4·5H2O, 0.248 g of Co(NO3)2·6H2O, and 0.114 g of H3BO3. The trace solution for P. denitrificans contained (per 100 mL) 1.5 g of Na2MoO4·2H2O, 0.04 g of CuSO4·5H2O, and ammonium Fe(III) citrate. The carbon source used for growth was 0.5% (w/v) (46 mM) n-BA, 20 mM choline chloride, or 60 mM glucose.
Antibiotics were supplied as needed at the following concentrations: 50 μg/mL ampicillin, 20 μg/mL rifampicin, 10 μg/mL (for E. coli) or 1 μg/mL (for R. sphaeroides and P. denitrificans) tetracycline, and 50 μg/mL streptomycin.
Construction of expression plasmids
Expression plasmids for qhp genes were constructed using a broad host range vector pRK415-1 (Keen et al. 1988), mostly using the standard molecular genetic methods and according to the following procedure:
(i) The NdeI/BamHI fragment excised from pRK-PA800-qhpG (Nakai et al. 2014) was inserted into pET-11a (Merck Millipore) to yield pET-qhpG. (ii) The EcoRI/BamHI fragment excised from pRK-Pweak-lacZ (Nakai et al. 2014) was inserted into pBBR-ORF2His (Nakai et al. 2015) (replacing qhpD) to yield pBBR-Pweak-lacZ. (iii) The AflIII/EcoRI fragment excised from pET-qhpG was inserted into pBBR-ORF2His (Nakai et al. 2015) to yield pBBR-qhpG-qhpD-H6. (iv) The EcoRI/NdeI fragment excised from pBBR-qhpG-qhpD-H6 was inserted into pBBR-Pweak-lacZ to yield pBBR-qhpG-lacZ. (v) The NdeI/BamHI fragment excised from pRK-PA800-qhpF (Nakai et al. 2014) was inserted into pBBR-qhpD (Nakai et al. 2015) (replacing qhpD) to yield pBBR-qhpF. (vi) The NdeI/StuI fragment containing qhpADC excised from pDS208 (Ono et al. 2006) and the StuI/NheI fragment containing qhpCBEF excised from pUC18-PstI (Datta et al. 2001) were inserted into pBBR-qhpF to yield pBBR-qhpADCBEF. (vii) The NdeI/SphI fragment excised from pBBR-qhpADCBEF was inserted into pBBR-qhpG-lacZ (replacing lacZ) to yield pBBR-qhpGADCBEF. (viii) pRK-PR600C-qhpR (Nakai et al. 2014) was digested with NdeI, blunt-ended using Klenow fragment and self-ligated to yield pRK-PR600C-qhpRΔNdeI. (ix) The BamHI fragment excised from pBBR-qhpGADCBEF was inserted into pRK-PR600C-qhpRΔNdeI to yield the final expression plasmid pRK-qhpGADCBEFR.
Expression and purification of QHNDH
The native QHNDH was expressed and purified essentially as described previously with minor modifications (Takagi et al. 1999). P. denitrificans was grown aerobically at 30 °C in the minimal medium (4 L) supplemented with 46 mM n-BA and 20 mM choline chloride. After 36 h, the cells were harvested by centrifugation, and the periplasmic fraction was prepared as described previously (Ono et al. 2006). Subsequent purification procedures were all performed on ice or at 4 °C. The periplasmic fraction was loaded on a 5-mL HiTrap Q column (Cytiva) equilibrated with 150 mM NaCl and 20 mM Tris-HCl (pH 8.0). Protein was eluted with a linear gradient of 0.15–1.0 M NaCl in the same buffer. Ammonium sulfate was added to the resulting eluate to a final concentration of 1.2 M, then the solution was applied to a Resource PHE column (6 mL) equilibrated with 50 mM sodium phosphate (pH 7.0) and 1.2 M ammonium sulfate. Protein was eluted with a linear gradient of 1.2–0 M ammonium sulfate in 50 mM sodium phosphate (pH 7.0). Fractions containing the objective protein colored reddish brown were collected and desalted by ultrafiltration with a 10 kDa cut-off centrifugal filter device (Amicon Ultra-4, Millipore). The resulting solution was applied to a 1-mL Resource Q column (Cytiva) equilibrated with 150 mM NaCl and 20 mM Tris-HCl (pH 8.0). Protein was eluted with a linear gradient of 0.15–1.0 M NaCl in the same buffer. Fractions containing the objective protein were then loaded onto a 120-mL Sephacryl S-200 column (Cytiva) equilibrated with 20 mM Tris-HCl buffer (pH 8.0) and 150 mM NaCl and eluted with the same buffer. The purified QHNDH was converted to the oxidized form by incubation with 1 mM potassium ferricyanide, and the buffer was exchanged for 20 mM Tris-HCl buffer (pH 8.0) and 150 mM NaCl by ultrafiltration. The purified protein was stored at −80 °C until use.
The QHNDH-expression plasmid (pRK-qhpGADCBEFR) was introduced into R. sphaeroides by diparental mating using E. coli S17-1 as the donor cells. R. sphaeroides cells harboring the expression plasmid were selected on a minimal medium plate (1.5% agar) containing 1 μg/mL tetracycline. The cells were grown aerobically at 30 °C in the minimal mineral medium (4 L) supplemented with 60 mM glucose and 1 μg/mL tetracycline. When the cell density reached an OD600 of 0.7–1.0, n-BA (final conc., 18 mM) was added to the culture medium and the cells were further grown for 36 h. The cells were harvested by centrifugation, and the enzyme was purified as described above except for an additional chromatography step: the eluate from the S-200 column was desalted by ultrafiltration, loaded on a 2-mL hydroxyapatite column CHT2-I (BioRad) equilibrated with 10 mM sodium phosphate (pH 7.0), and the protein was eluted with a linear gradient of 10–500 mM sodium phosphate (pH 7.0).
Mass spectrometric analysis
To obtain the γ-subunit that is heat-resistant, the purified QHNDH in 10 mM sodium phosphate (pH 7.0) was incubated with 1 mM tris(2-carboxyethyl)phosphine (TCEP) at 95 °C for 10 min (to denature the α- and β-subunits). To 10 μL of the solution was added 1 μL of 500 mM 2-iodoacetamide in 50 mM potassium phosphate (pH 7.5), and the mixture was kept at room temperature for 1 h. The mixture was acidified with 2% (v/v) formic acid, desalted with a C18 ZipTip pipette tip (Millipore), and eluted with 10 μL of 50% (v/v) acetonitrile in 0.1% (v/v) trifluoroacetic acid. The eluate (1 μL) was subjected to mass spectrometric analysis using a Bruker Ultraflex III MALDI-TOF mass spectrometer and 1 mg/mL sinapic acid (Bruker) dissolved in 90% (v/v) acetonitrile containing 0.1% (v/v) trifluoroacetic acid, as a matrix, which was co-crystallized with the protein by the drying-droplet method. Before every mass spectrometric analysis, mass calibration was performed using a Protein Calibration Standard I (Bruker).
Other methods
Preparation of periplasmic fractions of P. denitrificans and R. sphaeroides cells, QHNDH activity and protein assays, SDS-PAGE, Western blotting, and quinone staining were performed as described previously (Ono et al. 2006; Nakai et al. 2012; Nakai et al. 2014). QHNDH concentration was determined spectrophotometrically using the extinction coefficient of 227,000 M−1 cm−1 at 408 nm (Takagi et al. 1999). 1 U of the enzyme activity was defined as the amount that reduces 1 µmol of 2,6-dichlorophenolindophenol per min at 25 °C (Ono et al. 2006).
Supplementary Figure
Figure S1
Growth of non-transformed R. sphaeroides (Rsp) in the minimal medium containing n-butyraldehyde or n-butyric acid. Cells of Rsp were grown in the minimal medium containing 0.10% n-butyric acid (■), or 0.50% (♦), 0.10% (○), and 0.05% (+) n-butyraldehyde. To reduce the toxicity of n-butyraldehyde, 0.02% of n-butyraldehyde was added five times to the medium at 0, 36, 48, 60, and 72 h (0.10% in total) (▲). Cell densities measured by OD600 were plotted against culture time (h).
Supplementary References
Datta S, Mori Y, Takagi K et al. Structure of a quinohemoprotein amine dehydrogenase with an uncommon redox cofactor and highly unusual crosslinking. Proc Natl Acad Sci USA 2001;98:14268–73.
Keen NT, Tamaki S, Kobayashi D et al. Improved broad-host-range plasmids for DNA cloning in gram-negative bacteria. Gene 1988;70:191–7.
Nakai T, Deguchi T, Frebort I et al. Identification of genes essential for the biogenesis of quinohemoprotein amine dehydrogenase. Biochemistry 2014;53:895–907.
Nakai T, Ito H, Kobayashi K et al. The radical S-adenosyl-L-methionine enzyme QhpD catalyzes sequential formation of intra-protein sulfur-to-methylene carbon thioether bonds, J Biol Chem 2015;290:11144–66.
Nakai T, Ono K, Kuroda S et al. An unusual subtilisin-like serine protease is essential for biogenesis of quinohemoprotein amine dehydrogenase. J Biol Chem 2012;287:6530–8.
Ono K, Okajima T, Tani M et al. Involvement of a putative [Fe-S]-cluster-binding protein in the biogenesis of quinohemoprotein amine dehydrogenase. J Biol Chem 2006;281:13672–84.
Takagi K, Torimura M, Kawaguchi K et al. Biochemical and electrochemical characterization of quinohemoprotein amine dehydrogenase from Paracoccus denitrificans. Biochemistry 1999;38:6935–42.
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