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Glutathione-Dependent Formaldehyde Dehydrogenase Homolog from Bacillus subtilis Strain R5 is a Propanol-Preferring Alcohol Dehydrogenase

Raza Ashraf1, Naeem Rashid1*, Saadia Basheer1, Iram Aziz1, and Muhammad Akhtar2

1School of Biological Sciences, University of the Punjab, Quaid-e-Azam Campus, Lahore 54590, Pakistan; E-mail: naeem.ff.sbs@pu.edu.pk, naeemrashid37@hotmail.com

2School of Biological Sciences, University of Southampton, Southampton SO16 7PX, UK

* To whom correspondence should be addressed.

Received July 2, 2016; Revision received August 26, 2016
Genome search of Bacillus subtilis revealed the presence of an open reading frame annotated as glutathione-dependent formaldehyde dehydrogenase/alcohol dehydrogenase. The open reading frame consists of 1137 nucleotides corresponding to a polypeptide of 378 amino acids. To examine whether the encoded protein is glutathione-dependent formaldehyde dehydrogenase or alcohol dehydrogenase, we cloned and characterized the gene product. Enzyme activity assays revealed that the enzyme exhibits a metal ion-dependent alcohol dehydrogenase activity but no glutathione-dependent formaldehyde dehydrogenase or aldehyde dismutase activity. Although the protein is of mesophilic origin, optimal temperature for the enzyme activity is 60°C. Thermostability analysis by circular dichroism spectroscopy revealed that the protein is stable up to 60°C. Presence or absence of metal ions in the reaction mixture did not affect the enzyme activity. However, metal ions were necessary at the time of protein production and folding. There was a marked difference in the enzyme activity and CD spectra of the proteins produced in the presence and absence of metal ions. The experimental results obtained in this study demonstrate that the enzyme is a bona-fide alcohol dehydrogenase and not a glutathione-dependent formaldehyde dehydrogenase.
KEY WORDS: Bacillus subtilis, formaldehyde dehydrogenase, alcohol dehydrogenase, metal dependent, protein folding, circular dichroism

DOI: 10.1134/S0006297917010023