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J. Biol. Chem., Vol. 276, Issue 20, 17429-17436, May 18, 2001
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From the Department of Microbiology, University of Illinois,
Chemical and Life Sciences Laboratory, Urbana, Illinois 61801
The ptxD gene from
Pseudomonas stutzeri WM88 encoding the novel phosphorus
oxidizing enzyme NAD:phosphite oxidoreductase (trivial name phosphite
dehydrogenase, PtxD) was cloned into an expression vector and
overproduced in Escherichia coli. The heterologously produced enzyme is indistinguishable from the native enzyme based on
mass spectrometry, amino-terminal sequencing, and specific activity
analyses. Recombinant PtxD was purified to homogeneity via a two-step
affinity protocol and characterized. The enzyme stoichiometrically
produces NADH and phosphate from NAD and phosphite. The reverse
reaction was not observed. Gel filtration analysis of the purified
protein is consistent with PtxD acting as a homodimer. PtxD has a high
affinity for its substrates with Km values of
53.1 ± 6.7 µM and 54.6 ± 6.7 µM, for phosphite and NAD, respectively. Vmax and kcat were
determined to be 12.2 ± 0.3 µmol min
Purification and Characterization of a Novel Phosphorus-oxidizing
Enzyme from Pseudomonas stutzeri WM88*,
,
1
mg1 and 440 min1.
NADP can substitute poorly for NAD; however, none of the numerous compounds examined were able to substitute for phosphite. Initial rate
studies in the absence or presence of products and in the presence of
the dead end inhibitor sulfite are most consistent with a sequential
ordered mechanism for the PtxD reaction, with NAD binding first and
NADH being released last. Amino acid sequence comparisons place PtxD as
a new member of the D-2-hydroxyacid NAD-dependent dehydrogenases, the only one to have an
inorganic substrate. To our knowledge, this is the first detailed
biochemical study on an enzyme capable of direct oxidation of a reduced
phosphorus compound.
*
This work was supported in part by National Institutes of
Health Grant GM59334. The Voyager mass spectrometer used by the University of Illinois Mass Spectrometry facility was purchased in part
with Division of Research Resources, National Institutes of Health,
Grant RR 11966.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org) contains six tables.
Supported by a DeBeor Fellowship from the University of Illinois
Department of Microbiology.
§
Supported by National Institutes of Health Grant GM07283-26.
¶
To whom correspondence should be addressed: Dept. of
Microbiology, University of Illinois, B103 Chemical and Life Sciences Laboratory, 601 S. Goodwin, Urbana, IL 61801. Tel.: 217-244-1943; Fax:
217-244-6697; E-mail: metcalf@uiuc.edu.
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