Cloning, expression, and characterization of an aldehyde dehydrogenase from Escherichia coli K-12 that utilizes 3-Hydroxypropionaldehyde as a substrate

  • Ji-Eun Jo
  • Subramanian Mohan Raj
  • Chelladurai Rathnasingh
  • Edwardraja Selvakumar
  • Woo-Chel Jung
  • Sunghoon ParkEmail author
Biotechnologically Relevant Enzymes and Proteins


3-Hydroxypropionaldehyde (3-HPA), an intermediary compound of glycerol metabolism in bacteria, serves as a precursor to 3-Hydroxypropionic acid (3-HP), a commercially valuable platform chemical. To achieve the effective conversion of 3-HPA to 3-HP, an aldH gene encoding an aldehyde dehydrogenase in Escherichia coli K-12 (AldH) was cloned, expressed, and characterized for its properties. The recombinant AldH exhibited broad substrate specificity for various aliphatic and aromatic aldehydes. AldH preferred NAD+ over NADP+ as a cofactor for the oxidation of most aliphatic aldehydes tested. Among the aldehydes used, the specific activity was highest (38.1 U mg−1 protein) for 3-HPA at pH 8.0 and 37 °C. The catalytic efficiency (k cat) and the specificity constant (k cat/K m) for 3-HPA in the presence of NAD+ were 28.5 s−1 and 58.6 × 103 M−1 s−1, respectively. The AldH activity was enhanced in the presence of disulfide reductants such as dithiothreitol (DTT) or 2-mercaptoethanol, while several metal ions, particularly Hg2+, Ag+, Cu2+, and Zn2+, inhibited AldH activity. This study illustrates that AldH is a potentially useful enzyme in converting 3-HPA to 3-HP.


Aldehyde dehydrogenase aldH 3-Hydroxypropionaldehyde dehydrogenase 3-Hydroxypropionic acid Enzyme characterization Escherichia coli 



This work was financially supported by the Korean Ministry of Commerce, Industry, and Energy (Grant No. 10028380-2006-11). Dr. SM Raj is grateful to the Brain Korea 21 program (Pusan National University) for financial assistance. The authors wish to express their gratitude to Prof. Watanabe (Kyoto University, Japan) for providing the pQE-80L vector.


  1. Baldoma L, Aguilar J (1987) Involvement of lactaldehyde dehydrogenase in several metabolic pathways of Escherichia coli K-12. J Biol Chem 262:13991–13996Google Scholar
  2. Bradford MM (1976) A rapid and sensitive for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefGoogle Scholar
  3. Branden C, Tooze J (1991) Introduction to protein structure, 2nd edn. Garland, New YorkGoogle Scholar
  4. Fuhrer T, Chen L, Sauer U, Vitkup D (2007) Computational prediction and experimental verification of the gene encoding the NAD+/NADP+-dependent succinate semialdehyde dehydrogenase in Escherichia coli. J Bacteriol 189:8073–8078CrossRefGoogle Scholar
  5. Gruez A, Roig-Zamboni V, Salomoni SGA, Valencia C, Tegoni VCM, Cambillau C (2004) Crystal structure and kinetics identify Escherichia coli ydcW gene product as a medium-chain aldehyde dehydrogenase. J Mol Biol 343:29–41CrossRefGoogle Scholar
  6. Hall RH, Stern ES (1950) Acid-catalysed hydration of acrylaldehyde: kinetics of the reaction and isolation of b-hydroxypropionaldehyde. J Chem Soc 1950:490–498CrossRefGoogle Scholar
  7. Hayashi K, Morooka N, Yamamoto Y, Fujita K, Isono K, Chio S, Ohtsubo E, Baba T, Wanner BL, Mori H, Horiuchi T (2006) Highly accurate genome sequences of Escherichia coli K-12 strains MG1665 and W3110. Mol Syst Biol 2:7CrossRefGoogle Scholar
  8. Heim R, Strehler EE (1991) Cloning an Escherichia coli gene encoding a protein remarkably similar to mammalian aldehyde dehydrogenases. Gene 99:15–23CrossRefGoogle Scholar
  9. Hempel J, Nicholas H, Lindahl R (1993) Aldehyde dehydrogenases: widespread structural and functional diversity within a shared framework. Protein Sci 2:1890–1900Google Scholar
  10. Hidalgo E, Chen Lin YECC, Aguilar J (1991) Molecular cloning and DNA sequencing of the Escherichia coli K-12 ald gene encoding aldehyde dehydrogenase. J Bacteriol 173:6118–6123Google Scholar
  11. Ho KK, Weiner H (2005) Isolation and characterization of an aldehyde dehydrogenase encoded by the aldB gene of Escherichia coli. J Bacteriol 187:1067–1073CrossRefGoogle Scholar
  12. Jaureguibeitia A, Saa L, Llama MJ, Serra JL (2007) Purification, characterization and cloning of aldehyde dehydrogenase from Rhodococcus erythropolis UPV-1. Appl Microbiol Biotechnol 73:1073–1086CrossRefGoogle Scholar
  13. Kim HG, Kim Y, Lim HM, Shin HJ, Kim SW (2006) Purification, characterization, and cloning of trimethylamine dehydrogenase from Methylophaga sp. strain SK1. Biotechnol Bioprocess Engg 11:337–343CrossRefGoogle Scholar
  14. Kurihara S, Oda S, Kato K, Kim HG, Koyanagi T, Kumagai H, Suzuki H (2005) A novel putrescine utilization pathway involves γ-glutamylated intermediates of Escherichia coli K-12. J Biol Chem 280:4602–4608CrossRefGoogle Scholar
  15. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefGoogle Scholar
  16. Leal NA, Havemann GD, Bobik TA (2003) PduP is a coenzyme-A-acylating propionaldehyde dehydrogenase associated with the polyhedral bodies involved in B12-dependent 1,2-propanediol degradation by Salmonella enterica serovar Typhimurium LT2. Arch Microbiol 180:353–361CrossRefGoogle Scholar
  17. Limon A, Hidalgo E, Aguilar J (1997) The aldA gene of Escherichia coli is under the control of at least three transcriptional regulators. Microbiol 143:2085–2095Google Scholar
  18. Membrillo-Hernandez J, Echave P, Cabiscol E, Tamarit J, Ros J, Lin ECC (2000) Evolution of the adhE gene product of Escherichia coli from a functional reductase to a dehydrogenase. J Biol Chem 275:33869–33875CrossRefGoogle Scholar
  19. Mori N, Yoshida N, Kitamoto Y (1992) Purification and properties of betaine aldehyde dehydrogenase from Xanthomonas translucens. J Ferment Bioeng 73:352–356CrossRefGoogle Scholar
  20. Nagasawa T, Kawabata Y, Tani Y, Ogata K (1976) Purification and characterization of betaine aldehyde dehydrogenase from Pseudomonas aeruginosa A-16. Agric Biol Chem 40:1743–1749Google Scholar
  21. Ohta T, Tani A, Kimbara K, Kawai F (2005) A novel nicotinoprotein aldehyde dehydrogenase involved in polyethylene glycol degradation. Appl Microbiol Biotechnol 68:639–646CrossRefGoogle Scholar
  22. Perozich J, Nicholas H, Wang BC, Lindahl R, Hempel J (1999) Relationships within the aldehyde dehydrogenase extended family. Protein Sci 8:137–146Google Scholar
  23. Perozich J, Kuo I, Wang BC, Boesch JS, Lindahl R, Hempel J (2000) Shifting the NAD/NADP preference in class 3 aldehyde dehydrogenase. Eur J Biochem 267:6197–6203CrossRefGoogle Scholar
  24. Perozich J, Kuo I, Lindahl R, Hempel J (2001) Coenzyme specificity in aldehyde dehydrogenase. Chem Biol Interactions 130–132:115–124CrossRefGoogle Scholar
  25. Prieto MA, Diaz E, Garcia JL (1996) Molecular characterization of the 4-hydroxyphenylacetate catabolic pathway of Escherichia coli W: engineering a mobile aromatic degradative cluster. J Bacteriol 178:111–120CrossRefGoogle Scholar
  26. Sambrook J, Russell D (2001) Molecular cloning—a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  27. Savoly A, Elko DP, Veal B, McMahon ML, Heller DB (2005) Patent No. 6,863,837Google Scholar
  28. Sophos NA, Vasiliou V (2003) Aldehyde dehydrogenase gene superfamily: the 2002 update. Chem Biol Interactions 143–144:5–22CrossRefGoogle Scholar
  29. Steinman CR, Jakoby WB (1967) Yeast aldehyde dehydrogenase. J Biol Chem 242:5019–5023Google Scholar
  30. Tigerstrom RGV, Razzell WE (1968) Aldehyde dehydrogenase. J Biol Chem 243:2691–2702Google Scholar
  31. Ting HH, Crabbe MJ (1983) Bovine lens aldehyde dehydrogenase. Kinetics and mechanism. J Biochem 2:361–368Google Scholar
  32. Toth J, Ismaiel AA, Chen JS (1999) The ald gene, encoding a coenzyme A-acylating aldehyde dehydrogenase, distinguishes Clostridium beijerinckii and two other solvent-producing Clostridia form Clostridium acetobutylicum. Appl Environ Microbiol 65:4973–4980Google Scholar
  33. Velasco-Garcia R, Mujica-Jimenez C, Mendoza-Hernandez G, Munoz-Clares RA (1999) Rapid purification and properties of betaine aldehyde dehydrogenase from Pseudomonas aeruginosa. J Bacteriol 181:1292–1300Google Scholar
  34. Watanabe S, Yamada M, Ohtsu I, Makino K (2007) α-ketoglutaric semialdehyde dehydrogenase isozymes involved in metabolic pathways of D-glucarate, D-galactarate, and hydroxy-L-proline. J Biol Chem 282:6685–6695CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Ji-Eun Jo
    • 1
  • Subramanian Mohan Raj
    • 1
  • Chelladurai Rathnasingh
    • 1
  • Edwardraja Selvakumar
    • 1
  • Woo-Chel Jung
    • 1
  • Sunghoon Park
    • 1
    Email author
  1. 1.Department of Chemical and Biochemical EngineeringPusan National UniversityBusanRepublic of Korea

Personalised recommendations