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PMID:23828599

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Citation

Zeiser, J, Mühlenbeck, LH, Schweiger, P and Deppenmeier, U' (2013) Characterization of a periplasmic quinoprotein from Sphingomonas wittichii that functions as aldehyde dehydrogenase. Appl. Microbiol. Biotechnol. '

Abstract

The α-proteobacterium Sphingomonas wittichii RW1 is known for its ability to degrade dioxins and related toxic substances. Bioinformatic analysis of the genome indicated that this organism may contain the largest number of pyrroloquinoline quinone-dependent dehydrogenases of any bacteria sequenced so far. Sequence analysis also showed that one of these genes (swit_4395) encodes an enzyme that belongs to the class of periplasmic glucose dehydrogenases. This gene was fused to a pelB signal sequence and a strep-tag coding region at the 5' and 3' ends, respectively. The fusion product was cloned into the broad-host range expression vector pBBR1p264-Streplong and the corresponding protein was heterologously produced in Escherichia coli, purified via Strep-Tactin affinity chromatography, and characterized. The protein Swit_4395 had a subunit mass of 39.3 kDa and formed active homooctamers and homododecamers. The enzyme showed the highest activities with short- and medium-chain aldehydes (chain length C1-C6) and ketoaldehydes, such as methylglyoxal and phenylglyoxal. Butyraldehyde was the best substrate, with V max and apparent K M values of 3,970 U/mg protein and 12.3 mM, respectively. Pyrroloquinoline quinone was detected using UV-Vis spectroscopy and was found to be a prosthetic group of the purified enzyme. Therefore, Swit_4395 was identified as a pyrroloquinoline quinone-dependent aldehyde dehydrogenase. The enzyme could be purified from the native host when the expression vector was introduced into S. wittichii RW1, indicating homologous protein production. Overproduction of Swit_4395 in S. wittichii RW1 dramatically increased the tolerance of the bacterium toward butyraldehyde and thus might contribute to the detoxification of toxic aldehydes.

Links

PubMed Online version:10.1007/s00253-013-5016-5

Keywords


Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

SPHWW:A5VEL7

GO:0046185: aldehyde catabolic process

ECO:0000315:

P

Fig 3 showing growth of Swit_4395 mutants w/i concentrations of butyraldehyde.

complete
CACAO 8540

SPHWW:A5VEL7

GO:0047113: aldehyde dehydrogenase (pyrroloquinoline-quinone) activity

ECO:0000314:

F

Table 2

complete
CACAO 8555

SPHWW:A5VEL7

enables

GO:0047113: aldehyde dehydrogenase (quinone) activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

SPHWW:A5VEL7

involved_in

GO:0046185: aldehyde catabolic process

ECO:0000315: mutant phenotype evidence used in manual assertion

P

Seeded From UniProt

complete

See also

References

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