A previous study of the effect of zinc deprivation on Mycobacterium bovis BCG pointed out the potential importance of an alcohol dehydrogenase for maintaining the hydrophobic character of the cell envelope. In this report, the effect of the overexpression of the M. bovis BCG alcohol dehydrogenase (ADH) in Mycobacterium smegmatis and M. bovis BCG is described. The purification of the enzyme was performed to apparent homogeneity from overexpressing M. bovis BCG cells and its kinetic parameters were determined. The enzyme showed a strong preference for both aromatic and aliphatic aldehydes while the corresponding alcohols were processed 100±1000-fold less efficiently. The best k cat /K m values were found with benzaldehyde . 3-methoxybenzaldehyde . octanal . coniferaldehyde. A phylogenetic analysis clearly revealed that the M. bovis BCG ADH together with the ADHs from Bacillus subtilis and Helicobacter pylori formed a sister group of the class C medium-chain alcohol dehydrogenases, the plant cinnamyl alcohol dehydrogenases (CADs). Comparison of the kinetic properties of our ADH with some related class C enzymes indicated that the mycobacterial enzyme substrate profile resembled that of the CADs involved in plant defence rather than those implicated in lignification. A possible role for the M. bovis BCG ADH in the biosynthesis of the lipids composing the mycobacterial cell envelope is proposed.Keywords: Mycobacterium bovis BCG; alcohol dehydrogenase; cinnamyl alcohol dehydrogenase.Alcohol dehydrogenases (ADHs) display a wide range of substrate specificities and fulfil several key physiological functions. They can be divided into three major categories. The first category is composed of the NAD(P)-dependent ADHs subdivided into three subgroups according to their metal dependence: (a) the medium-chain zinc-dependent enzymes; (b) the short-chain zinc-independent enzymes; and (c) the iron-activated enzymes. The NAD(P)-independent enzymes form the second category and the third category contains the oxidases which catalyse an essentially irreversible oxidation of alcohols [1].The vaccine strain Mycobacterium bovis BCG grows poorly on zinc-deprived Sauton medium, and forms a thin, pale, unfolded pellicle that gets wet and often sinks. This phenotype suggested a cell envelope with a reduced hydrophobic content. The cell composition and the products excreted into the medium, including aldehydes, can, to some extent, be correlated with a decrease of the specific activity of a soluble zinc-dependent alcohol dehydrogenase. This enzyme was partially purified from BCG cells and characterized as a dimeric NADP-dependent protein of broad specificity with higher affinities towards aldehydes than towards alcohols [2,3].The corresponding gene has been cloned and sequenced [4]. Comparison with other ADHs allowed classification of this enzyme into the zinc-containing, medium-chain alcohol/polyol dehydrogenase family which had been primarily described in eukaryotes [5]. Currently, this family is composed of the tetrameric and the dimeric...
Heat-shock proteins (Hsps) from various origins are known to share a conserved structure and are assumed to be key partners in the biogenesis of proteins. Fractionation of the mycobacterial Hsp60, a 65 kDa protein also called Cpn60, from Mycobacterium bovis BCG zinc-deficient culture filtrate on phenyl-Sepharose followed by Western blotting revealed the existence of four Hsp60-1 and Hsp60-2 forms, based on their hydrophobicity behaviour. Hsp60-2 species were further purified by ion-exchange chromatography and partial amino acid sequences of cyanogen bromide (CNBr) peptides of purified Hsp60-2 species showed identity with the amino acid sequence deduced from the hsp60-2 gene, indicating that the various Hsp60-2 forms are encoded by the same gene. In addition, the mycobacterial Hsp60-2 was overexpressed in E. coli using the pRR3Hsp60-2 plasmid and analysed on phenyl-Sepharose. The elution pattern of the recombinant Hsp60-2, as well as that of Escherichia coli GroEL, was similar to that of the native Hsp60-2 from the culture filtrate of M. bovis BCG and entirely different from that of the mycobacterial antigen 85. Extraction of mycobacterial Hsp60-2 forms, recombinant BCG Hsp60-2 and E. coli GroEL with organic solvents releases various amounts of non-covalently bound lipids. The presence of lipids on Hsp60-2 was confirmed by labelling M. bovis BCG with radioactive palmitate. The radioactivity was specifically associated with Hsp60 in the aqueous phase and the 19 and 38 kDa lipoproteins in the Triton X-114 phase. Analysis of the lipids extracted from purified Hsp60-2, recombinant BCG Hsp60-2 and E. coli GroEL by TLC showed the same pattern for all the samples. Acid methanolysis of the lipids followed by GC analysis led to the identification of C 16 :0 , C 18 :0 and C 18 :1 as the major fatty acyl constituents, and of methylglycoside in these proteins. Altogether, these data demonstrate that lipids are non-covalently bound to Hsp60-2 and homologous proteins.
The gene encoding of an alcohol dehydrogenase C (ADHC) from Mycobacterium smegmatis was cloned and sequenced. The protein encoded by this gene has 78% identity with Mycobacterium tuberculosis and Mycobacterium bovis BCG ADHC. The M. smegmatis ADHC was purified from M. smegmatis and the kinetic parameters of this enzyme showed that using NADPH as electron donor it has a strong preference for aliphatic and aromatic aldehyde substrates. Like the M. bovis BCG ADHC, this enzyme is more likely to act as an aldehyde reductase than as an alcohol dehydrogenase. The discovery of such an ADHC in a fast-growing, and easily engineered mycobacterial species opens the way to the utilisation of this M. smegmatis enzyme as a convenient model for the study of the physiological role of this alcohol dehydrogenase in mycobacteria.
S 0 3 7 8 -1 0 9 7 ( 0 1 ) 0 0 1 8 1 -1 Fig. 2. Alignment of mycobacterial ADHC amino acid sequences, deduced from the available genomic sequences of some mycobacteria, with Ms-ADHC. Identical or similar amino acids are in black or grey boxes, respectively. The region containing the characteristic zinc-dependent alcohol dehydrogenase signature is framed. The asterisks indicate the catalytic and structural zinc-binding residues. The ADHC sequences from M. tuberculosis (M. tub) and M. bovis BGC are 100% identical, as well as the sequences from M. avium and M. paratuberculosis (M. paratub.).
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