Purification and Characterization of a Microbial Dehydrogenase

Baré, Ghislain; Swiatkowski, Thierry; Moukil, Ahmed; Gerday, Charles; Thonart, Philippe

doi:10.1007/978-1-4612-0119-9_35

Cited by 3 publications

(3 citation statements)

References 20 publications

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“…However, the enzyme from strain BTP9 was a tetramer like that from strain TA1. It has been reported that the enzyme activity in strain BTP9 was not influenced by Mg 2+ or a chelating agent; however, the enzyme activity was approximately doubled in strain TA1 in the presence of Mg 2+ (Bare et al , 2002).…”

Section: Resultsmentioning

confidence: 99%

“…The characteristics of the purified enzyme were compared with another VDH purified from a previously isolated neutrophilic strain, Burkholderia cepacia TM1 (Tanaka & Hirokane, 2000). This report is an important contribution to the investigation of ferulic acid and vanillin metabolism because there have been only a few studies on the enzymatic characteristics of VDH (Bare et al , 2002), and it adds to the genetic information on VDH orthologs available in the bacterial genome databases.…”

Section: Introductionmentioning

confidence: 99%

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Purification and characterization of vanillin dehydrogenases from alkaliphileMicrococcussp. TA1 and neutrophileBurkholderia cepaciaTM1

Mitsui

Hirota

Tsuno

et al. 2010

FEMS Microbiology Letters

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Vanillin dehydrogenases (VDHs) were purified and characterized from two bacterial strains that have different pH dependencies for growth. The alkaliphile Micrococcus sp. TA1, isolated from an alkaline spa, can grow on several aromatic compounds such as ferulic acid, vanillin, vanillic acid, and protocatechuic acid under alkaline conditions. The neutrophile Burkholderia cepacia TM1, which was isolated previously, also grew on the above-mentioned compounds because they functioned as the sole carbon source under neutral conditions. Purified VDHs showed activities toward some aromatic aldehydes. These enzymes have the same subunit molecular mass of about 57 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but differed in some of their observed properties. Native molecular masses also differed between the purified enzymes. These were 250 kDa for the enzyme from alkaliphilic strain TA1 and 110 kDa for that from neutrophilic strain TM1, as determined by gel filtration. The enzyme from strain TA1 required NADP(+) as a coenzyme for its activity, but that from strain TM1 required NAD(+). These results are important because this is the first report of an alkaliphilic bacterium consuming lignin monomers.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Purification and characterization of vanillin dehydrogenases from alkaliphileMicrococcussp. TA1 and neutrophileBurkholderia cepaciaTM1

Mitsui

Hirota

Tsuno

et al. 2010

FEMS Microbiology Letters

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“…The enzyme assay was performed as described previously. 7,[37][38][39] Aldehyde dehydrogenase enzyme activity was monitored at 30 C using a ThermoScientic Multiskan™ Microplate spectrophotometer. The reactions contained 100 mM potassium phosphate buffer (pH 7.3), 0.5 mM NADP + , varied concentrations of substrates (10-1200 mM) and 0.6 mM enzyme.…”

Section: Aldh Activity Measurements and Enzyme Kinetic Analysismentioning

confidence: 99%

Characterization of Cd36_03230p, a putative vanillin dehydrogenase from Candida dubliniensis

2016

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A coding sequence (CD36-03230) from the yeast Candidadubliniensis had been previously annotated as a vanillin dehydrogenase (VDH). The corresponding protein (CD36-03230p) was recombinantly expressed in Escherichia coli and analysed. The protein is most likely a tetramer in solution as judged by crosslinking and gel filtration experiments. CD36-03230p is an active aldehyde dehydrogenase favouring cyclic and aromatic substrates. Positive cooperativity and substrate inhibition wereobserved with some substrates. The redox cofactor NADP+ andsubstrates affected the thermal stability of the protein. Interestingly, the enzyme had no detectable activity with vanillin suggesting that the annotation is incorrect. It has been previously hypothesized that a methionine residue at a key position in the active site of yeast aldehyde dehydrogenases sterically hinders cyclic substrates and restricts specificity to aliphatic aldehydes. Molecular modeling of CD36-03230p demonstrates that it has an isoleucine residue (Ile-156) at this position, further strengtheningthis hypothesis

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Purification and Characterization of a Microbial Dehydrogenase A Vanillin:NAD(P)<sup>+</sup> Oxidoreductase

Baré

Swiatkowski²,

Moukil³

et al. 2002

ABAB

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Pseudomonas fluorescens (strain BTP9) was found to have at least two NAD(P)-dependent vanillin dehydrogenases: one is induced by vanillin, and the other is constitutive. The constitutive enzyme was purified by ammonium sulfate fractionation, gel-filtration, and Q-Sepharose chromatography. The subunit Mr value was 55,000, determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The native Mr value estimated by gel-filtration chromatography gave a value of 210,000. The enzyme made use of NAD+ less effectively than NADP+. Benzaldehyde, 4-hydroxybenzaldehyde, hexanal, and acetaldehyde were not oxidized at detectable rates in the presence of NAD+ or NADP+. The ultraviolet absorption spectrum indicated that there is no cofactor or prosthetic group bound. The vanillin oxidation reaction was essentially irreversible. The pH optimum was 9.5 and the pI of the enzyme was 4.9. Enzyme activity was not affected when assayed in the presence of salts, except FeCl2. The enzyme was inhibited by the thiol-blocking reagents 4-chloromercuribenzoate and N-ethylmaleimide. NAD+ and NADP+ protected the enzyme against such a type of inhibition along with vanillin to a lesser extent. The enzyme exhibited esterase activity with 4-nitrophenyl acetate as substrate and was activated by low concentrations of NAD+ or NADP+. We compared the properties of the enzyme with those of some well-characterized microbial benzaldehyde dehydrogenases.

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Purification and Characterization of a Microbial Dehydrogenase

Cited by 3 publications

References 20 publications

Purification and characterization of vanillin dehydrogenases from alkaliphileMicrococcussp. TA1 and neutrophileBurkholderia cepaciaTM1

Purification and characterization of vanillin dehydrogenases from alkaliphileMicrococcussp. TA1 and neutrophileBurkholderia cepaciaTM1

Characterization of Cd36_03230p, a putative vanillin dehydrogenase from Candida dubliniensis

Purification and Characterization of a Microbial Dehydrogenase A Vanillin:NAD(P)<sup>+</sup> Oxidoreductase

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