Abstract
A gene (pegC) encoding aldehyde dehydrogenase (ALDH) was located 3.4 kb upstream of a gene encoding polyethylene glycol (PEG) dehydrogenase (pegA) in Sphingomonas macrogoltabidus strain 103. ALDH was expressed in Escherichia coli and purified on a Ni-nitrilotriacetic acid agarose column. The recombinant enzyme was a homotetramer consisting of four 46.1-kDa subunits. The alignment of the putative amino acid sequence of the cloned enzyme showed high similarity with a group of NAD(P)-dependent ALDHs (identity 36–52%); NAD-binding domains (Rossmann fold and four glycine residues) and catalytic residues (Glu225 and Cys259) were well conserved. The cofactor, which was extracted from the purified enzyme, was tightly bound to the enzyme and identified as NADP. The enzyme contained 0.94 mol NADP per subunit. The enzyme was activated by Ca2+, but by no other metals; no metal (Zn, Fe, Mg, or Mn) was detected in the purified recombinant enzyme. Activity was inhibited by p-chloromercuric benzoate, and heavy metals such as Hg, Cu, Pb and Cd, indicating that a cysteine residue is involved in the activity. Enzyme activity was independent of N,N-dimethyl-p-nitrosoaniline as an electron acceptor. Trans-4-(N,N-dimethylamino)-cinnamaldehyde was not oxidized as a substrate, but the compound worked as an inhibitor for the enzyme, as did pyrazole. The enzyme acted on n-aldehydes C2–C14) and PEG-aldehydes. Thus the enzyme was concluded to be a novel Ca2+-activating nicotinoprotein (NADP-containing) PEG-aldehyde dehydrogenase involved in the degradation of PEG in S. macrogoltabidus strain 103.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Arfman N, Hektor HJ, Bystrykh LV, Govorukhina NI, Dijkhuizen L, Frank J (1997) Properties of an NAD(H)-containing methanol dehydrogenase and its activator protein from Bacillus methanolicus. Eur J Biochem 244:426–433
Bystrykh LV, Govorukhina NI, van Ophem PW, Hektor HJ, Dijiikhuizen L, Duine JA (1993) Formaldehyde dismutase activities in Gram-positive bacteria oxidizing methanol. J Gen Microbiol 139:1979–1983
Farres J, Wang TTY, Cunningham SJ, Weiner H (1995) Investigation of the active site cysteine residue of rat liver mitochondrial aldehyde dehydrogenase by site-directed mutagenesis. Biochemistry 34:2592–2598
Hempel J, Perozich J, Wymore T, Nicholas HB (2002) An algorithm for identification and ranking of family-specific residues, applied to the ALDH3 family. Chem Biol Interact 143:23–28
Ito K (2002) Structural and functional analysis of enzymes and their application to clinical analysis—study on Pseudomonas putida formaldehyde dehydrogenase. Yakugaku Zasshi 122:805–811
Kaneko T, Nakamura Y, Sato S, Asamizu E, Kato T, Sasamoto S, Watanabe A, Idesawa K, Ishikawa A, Kawashima K, Kimura T, Kishida Y, Kiyokawa C, Kohara M, Matsumoto M, Matsuno A, Mochizuki Y, Nakayama S, Nakazaki N, Shimpo S, Sugimoto M, Takeuchi C, Yamada M, Tabata S (2000) Complete genome structure of the nitrogen-fixing symbiotic bacterium Mesorhizobium loti. DNA Res 7:331–338
Kawai F (2002) Microbial degradation of polyethers. Appl Microbiol Biotechnol 58:30–38
Kawai F, Yamanaka H (1986) Biodegradation of polyethylene glycol by symbiotic mixed culture (obligate mutualism). Arch Microbiol 146:125–129
Kawai F, Yamanaka H (1989) Inducible or constitutive polyethylene glycol dehydrogenase involved in the aerobic metabolism of polyethylene glycol. J Ferment Bioeng 67:300–302
Kawai F, Kimura T, Tani Y, Yamada H, Ueno T, Fukami H (1983) Identification of reaction products of polyethylene glycol dehydrogenase. Agric Biol Chem 47:1669–1671
Kleiger G, Eisenberg D (2002) GXXXG and GXXXA motifs stabilize FAD and NAD (P)-binding Rossmann folds through Cα–H..O hydrogen bonds and van der Waals interactions. J Mol Biol 323:69–76
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Lindahl R (1992) Aldehyde dehydrogenases and their role in carcinogenesis. Crit Rev Biochem Mol Biol 27:283–335
Navarro-avino JP, Prasad R, Miralles VJ, Benito RM, Serrano R (1999) A proposal for nomenclature of aldehyde dehydrogenases in Saccharomyces cerevisiae and characterization of the stress-inducible ALD2 and ALD3 genes. Yeast 15:829–842
Ophem PW van, Van Beeumen J, Duine JA (1993) Nicotinoprotein [NAD(P)-containing] alcohol/aldehyde oxidoreductases. Purification and characterization of a novel type from Amycolatopsis methanolica. Eur J Biochem 212:819–826
Perozich J, Nicholas H, Wang BC, Lidahl R, Hempel J (1999) Relationships within the aldehyde dehydrogenase extended family. Protein Sci 8:137–146
Piersma SR, Norin A, de Vries S, Jornvall H, Duine JA (2003) Inhibition of nicotinoprotein (NAD+-containing) alcohol dehydrogenase by trans-4-(N,N-dimethylamino) cinnamaldehyde binding to the active site. J Protein Chem 22:457–461
Pietruszko R, Lehmannm T, Dryjanski M, Abriola DP, Ambroziak W (2001) Catalysis of dehydrogenation of 4-trans(N,N-dimethylamino)cinnamaldehyde by aldehyde dehydrogenase. Chem Biol Interact 130–132:103–114
Pocard JA, Vincent N, Boncompagni E, Smith LT, Poggi MC, Rudulier DL (1997) Molecular characterization of the bet genes encoding glycine betaine synthesis in Sinorhizobium meliloti 102F34. Microbiology 143:1369–1379
Schenkels P, Duine JA (2000) Nicotinoprotein (NADH-containing) alcohol dehydrogenase from Rhodococcus erythropolis DSM 1069: an efficient catalyst for coenzyme-independent oxidation of a broad spectrum of alcohol and the interconversion of alcohols and aldehydes. Microbiology 146:775–785
Sheikh S, Ni L, Hurley TD, Weiner H (1997) The potential roles of the conserved amino acids in human liver mitochondrial aldehyde dehydrogenase. J Biol Chem 272:18817–18822
Stayner CK, Tweedie JW (1995) Cloning and characterisation of the cDNA for sheep liver cytosolic aldehyde dehydrogenase. Adv Exp Med Biol 372:61–66
Steinmetz CG, Xie PG, Weiner H, Hurley TD (1997) Structure of mitochondrial aldehyde dehydrogenase: the genetic component of ethanol aversion. Structure 5:701–711
Sugimoto M, Tanabe M, Hataya M, Enokibara S, Duine JA, Kawai F (2001) The first step in polyethylene glycol degradation by Sphingomonads proceed via flavoprotein alcohol dehydrogenase containing flavin adenine dinucleotide. J Bacteriol 183:6694–6698
Takeuchi M, Kawai F, Shimada Y, Yokota A (1993) Taxonomic study of polyethylene glycol-utilizing bacteria: emended description of the genes Sphingomonas and new description of Sphingomonas macrogoltabidus sp. nov., Sphingomonas sanguis sp. nov. and Sphingomonas terrae sp. nov. Syst Appl Microbiol 16:227–238
Tanaka N, Kusakabe Y, Ito K, Yoshimoto T, Nakamura K (2002) Crystal structure of formaldehyde dehydrogenase from Pseudomonas putida: the structural origin of the tightly bound cofactor in nicotinoprotein dehydrogenase. J Mol Biol 324:519–533
Weibel H, Hansen J (1989) Interaction of cinnamaldehyde (a sensitizer in fragrance) with protein. Contact Dermatitis 20:161–166
Yamanaka H, Kawai F (1989) Purification and characterization of constitutive polyethylene glycol (PEG) dehydrogenase of a PEG 4000-utilizing Flavobacterium sp. No. 203. J Ferment Bioeng 67:324–330
Yamashita M, Tani A, Kawai F (2004) A new ether bond-splitting enzyme found in Gram-positive polyethylene glycol 6000-utilizing bacterium, Pseudonocardia sp. strain K1. Appl Microbiol Biotechnol 66:174–179
Yanase H, Moriya K, Mukai N, Kawata Y, Okamoto K, Kato N (2002) Effects of GroESL coexpression on the folding of nicotinoprotein formaldehyde dismutase from Pseudomonas putida F61. Biosci Biotechnol Biochem 66:85–91
Yoshida A, Rzhetsky A, Hsu LC, Chang C (1998) Human aldehyde dehydrogenase gene family. Eur J Biochem 251:549–557
Acknowledgement
We are grateful to Mr. H. Nishizaki for atomic absorption analysis of the purified recombinant enzyme.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Ohta, T., Tani, A., Kimbara, K. et al. A novel nicotinoprotein aldehyde dehydrogenase involved in polyethylene glycol degradation. Appl Microbiol Biotechnol 68, 639–646 (2005). https://doi.org/10.1007/s00253-005-1936-z
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-005-1936-z