Molecular cloning and kinetic characterization of an aldehyde dehydrogenase gene in Klebsiella pneumoniae
Journal
Journal of the Formosan Medical Association
Journal Volume
104
Journal Issue
4
Pages
221-230
Date Issued
2005
Author(s)
Abstract
Background and Purpose: Klebsiella pneumoniae liver abscess with metastatic complications is an emerging infectious disease in Taiwan. The present study aimed to identify virulence genes involved in the pathogenicity of K. pneumoniae. Methods. The closely related Escherichia coli genome array was employed to study the expression of the putative genome of K. pneumoniae. Total mRNA expression levels of a K. pneumoniae strain (designated National Taiwan University Hospital [NTUH]-K2044), isolated from a patient with liver abscess, and another strain (designated NTUH-K9), from a patient with sepsis only, were compared on the E. coli array. RNA blot was used to reconfirm mRNA expression in NTUH-K9, K2044 and in 9 other sepsis strains and 9 other liver abcess strains. Results: One of the genes which was found to be highly expressed in NTUH-K2044, designated aldA, was selected for further study. The aldA gene codes for the enzyme aldehyde dehydrogenase (aldehyde:NAD[P]+ oxidoreductase; ALDH). Kinetic properties of ALDH isolated from the 2 strains, designated K2044 ALDH and K9 ALDH respectively, were characterized. The isolated recombinant K2044 ALDH and K9 ALDH, both with subunit molecular weight 55 kDa, exhibited similar substrate specificity and coenzyme preference with glycolaldehayde (Vmax/Km = 27 and 17 U/mg/mM, respectively) and glyceraldehyde (maximum velocity [Vmax]/Michaelis constant [K m] = 42 and 30 U/mg/mM, respectively) being the much better substrates and NAD+ being the preferred coenzyme (Km = 0.28 and 0.28 mM, respectively). Unlike K9 ALDH, K2044 ALDH displayed inhibition at high concentrations of glycolaldehyde (substrate inhibition constant [K 1] = 7.4 mM) and glyceraldehyde (K1 = 2.6 mM). Conclusion: The expression of the aldA gene is higher in K. pneumoniae strains from patients with liver access. The aldA gene encodes functional ALDH and can use glycolaldehyde and glyceraldehydes as substrates.
SDGs
Other Subjects
aldehyde dehydrogenase; glyceraldehyde; messenger RNA; nicotinamide adenine dinucleotide phosphate; oxidoreductase; recombinant enzyme; aldehyde dehydrogenase; astemizole; article; bacterial strain; controlled study; Escherichia coli; gene expression; genome; human; human tissue; kinetics; Klebsiella pneumoniae; molecular cloning; molecular weight; Northern blotting; nucleotide sequence; pathogenicity; sepsis; Taiwan; university hospital; amino acid sequence; Enterobacter infection; enzyme specificity; enzymology; genetics; Klebsiella pneumoniae; liver abscess; microbiology; molecular cloning; molecular genetics; nucleotide sequence; virulence; Aldehyde Dehydrogenase; Amino Acid Sequence; Astemizole; Base Sequence; Cloning, Molecular; Escherichia coli; Humans; Klebsiella Infections; Klebsiella pneumoniae; Liver Abscess; Molecular Sequence Data; Substrate Specificity; Virulence
Type
journal article