Butane-grown Pseudomonas butanovora oxidized butane by a soluble
butane monooxygenase through the terminal pathway yielding 1 -butanol as the
predominant product. Alcohol dehydrogenases (ADHs) involved in butane
oxidation in P. butanovora were purified and characterized at the biochemical,
genetic and physiological levels. Butane-grown P. butanovora expressed a type I
soluble quinoprotein 1 -butanol dehydrogenase (BOH), a soluble type II
quinohemoprotein 1 -butanol dehydrogenase (BDH) and an NAD���-dependent
secondary ADH. Two additional NAD���-dependent secondary ADHs were also
detected in cells grown on 2-butanol and lactate. BDH was purified to near
homogeneity and characterized. BDH is a monomer of 66 kDa consisting of one
mole of pyrroloquinoline quinone (PQQ) and 0.25 mole of heme c as the prosthetic
groups. BOH was partially purified and its deduced amino acid sequence suggests
a 67-kDa ADH containing a PQQ as a cofactor. BOH and BDH exhibited high
activities and preference towards I -butanol and fair preference towards
butyraldehyde. While BDH could not oxidize 2-butanol, BOH is capable of
2-butanol oxidation and has a broader substrate range than that of BDH. Genes
encoding BOH and BDH and their deduced amino acid sequences were identified.
BOH and BDH mRNAs and 1-butanol oxidation activity were induced when cells
were exposed to butane. Primary C��� and C��� alcohols were the most effective
inducers for boh and bdh. Some secondary alcohols, such as 2-butanol, were also
inducers for BOH mRNA, but not for BDH mRNA. Insertional inactivation of boh
or bdh affected unfavorably, but did not eliminate, butane utilization in
P. butanovora. The P. butanovora mutant strain with both boh and bdh genes
disrupted was unable to grow on butane and 1-butanol. This result confirmed the
involvement of BOH and BDH in butane and 1-butanol metabolism in
P. butanovora. Roles of B011 and BDH in butane and 1-butanol metabolism were
further studied at the physiological level. There are no substantial differences
between BOH and BDH in the mRNA expressions in response to three different 1-
butanol levels tested and in their abilities to respond to 1-butanol toxicity.
Different bioenergetic roles of BOH and BDH in butane and 1-butanol metabolism
were suggested. A model of 1 -butanol- dependent respiratory systems was
proposed where the electrons from 1 -butanol oxidation follow a branched electron
transport chain. The role of BOH was suggested to function primarily in energy
generation because B011 may couple to ubiquinone with the electrons being
transported to a cyanide-sensitive terminal oxidase. BDH may be more important
in the detoxification of 1 -butanol because the electrons from BDH may be
transferred to a terminal oxidase system that is less sensitive to cyanide, which is
not capable of energy generation. / Graduation date: 2003
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/31602 |
| Date | 17 May 2002 |
| Creators | Vangnai, Alisa S. |
| Contributors | Arp, Daniel J. |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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