Investigation of the role of Arg301 identified in the X-ray structure of phosphite dehydrogenase.

Biochemistry
John E HungWilfred A van der Donk

Abstract

Phosphite dehydrogenase (PTDH) from Pseudomonas stutzeri catalyzes the nicotinamide adenine dinucleotide-dependent oxidation of phosphite to phosphate. The enzyme belongs to the family of D-hydroxy acid dehydrogenases (DHDHs). A search of the protein databases uncovered many additional putative phosphite dehydrogenases. The genes encoding four diverse candidates were cloned and expressed, and the enzymes were purified and characterized. All oxidized phosphite to phosphate and had similar kinetic parameters despite a low level of pairwise sequence identity (39-72%). A recent crystal structure identified Arg301 as a residue in the active site that has not been investigated previously. Arg301 is fully conserved in the enzymes shown here to be PTDHs, but the residue is not conserved in other DHDHs. Kinetic analysis of site-directed mutants of this residue shows that it is important for efficient catalysis, with an ~100-fold decrease in k(cat) and an almost 700-fold increase in K(m,phosphite) for the R301A mutant. Interestingly, the R301K mutant displayed a slightly higher k(cat) than the parent PTDH, and a more modest increase in K(m) for phosphite (nearly 40-fold). Given these results, Arg301 may be involved in the binding and ori...Continue Reading

References

Nov 15, 1979·Analytical Biochemistry·P A LanzettaO A Candia
Jan 1, 1979·Methods in Enzymology·W W Cleland
Jan 1, 1982·Methods in Enzymology·K J Ellis, J F Morrison
Jun 15, 1999·The Plant Cell·R D ScavettaF Jurnak
Mar 30, 2001·The Journal of Biological Chemistry·A M CostasW W Metcalf
Jul 18, 2001·Journal of the American Chemical Society·J M VrtisW A van der Donk
Oct 9, 2002·Angewandte Chemie·Jennifer M VrtisWilfred A van der Donk
Sep 11, 2002·Proceedings of the National Academy of Sciences of the United States of America·Simon J CharnockGideon J Davies
Dec 8, 2004·Archives of Biochemistry and Biophysics·Yollete V Guillén Schlippe, Lizbeth Hedstrom
Mar 23, 2005·Biochemistry·Ryan WoodyerWilfred A van der Donk
Apr 27, 2005·Biochemistry·Heather A RelyeaWilfred A van der Donk
Sep 1, 2005·Biochemistry·Yollete V Guillén Schlippe, Lizbeth Hedstrom
Oct 6, 2005·Applied and Environmental Microbiology·Tyler W JohannesHuimin Zhao
Dec 14, 2005·Biochemistry·Yollete V Guillén Schlippe, Lizbeth Hedstrom
May 27, 2006·Combinatorial Chemistry & High Throughput Screening·Ryan WoodyerHuimin Zhao
Aug 22, 2006·Current Opinion in Chemical Biology·Lizbeth Hedstrom, Lu Gan
Sep 2, 2006·Biotechnology and Bioengineering·Tyler W JohannesHuimin Zhao
Aug 30, 2008·PLoS Biology·Donghong MinWei Yang
Oct 2, 2009·Chembiochem : a European Journal of Chemical Biology·Daniel E Torres PazmiñoMarco W Fraaije
Jan 13, 2011·Organic & Biomolecular Chemistry·Ana Rioz-MartínezMarco W Fraaije

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Citations

Oct 28, 2016·Chemical Reviews·Geoff P Horsman, David L Zechel
Jan 11, 2017·Chemical Science·Zedu HuangWilfred A van der Donk
Dec 12, 2018·Journal of the American Chemical Society·Graeme W Howe, Wilfred A van der Donk

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Methods Mentioned

BETA
X-ray
PCR
electrophoresis
gel filtration

Software Mentioned

OriginPro
ExPASy ProtParam
GraFit

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