Prospecting for unannotated enzymes

Discovery of a 3′,5′- nucleotide bisphosphate phosphatase within the amidohydrolase superfamily

Jennifer A. Cummings, Matthew Vetting, Swapnil V. Ghodge, Chengfu Xu, Brandan Hillerich, Ronald D. Seidel, Steven C. Almo, Frank M. Raushel

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

In bacteria, 3′,5′-adenosine bisphosphate (pAp) is generated from 3′-phosphoadenosine 5′-phosphosulfate in the sulfate assimilation pathway, and from coenzyme A by the transfer of the phosphopantetheine group to the acyl-carrier protein. pAp is subsequently hydrolyzed to 5′-AMP and orthophosphate, and this reaction has been shown to be important for superoxide stress tolerance. Herein, we report the discovery of the first instance of an enzyme from the amidohydrolase superfamily that is capable of hydrolyzing pAp. Crystal structures of Cv1693 from Chromobacterium violaceum have been determined to a resolution of 1.9 Å with AMP and orthophosphate bound in the active site. The enzyme has a trinuclear metal center in the active site with three Mn2+ ions. This enzyme (Cv1693) belongs to the Cluster of Orthologous Groups cog0613 from the polymerase and histidinol phosphatase family of enzymes. The values of kcat and kcat/Km for the hydrolysis of pAp are 22 s-1 and 1.4 × 106 M-1 s-1, respectively. The enzyme is promiscuous and is able to hydrolyze other 3′,5′- bisphosphonucleotides (pGp, pCp, pUp, and pIp) and 2′-deoxynucleotides with comparable catalytic efficiency. The enzyme is capable of hydrolyzing short oligonucleotides (pdA)5, albeit at rates much lower than that of pAp. Enzymes from two other enzyme families have previously been found to hydrolyze pAp at physiologically significant rates. These enzymes include CysQ from Escherichia coli (cog1218) and YtqI/NrnA from Bacillus subtilis (cog0618). Identification of the functional homologues to the experimentally verified pAp phosphatases from cog0613, cog1218, and cog0618 suggests that there is relatively little overlap of enzymes with this function in sequenced bacterial genomes.

Original languageEnglish (US)
Pages (from-to)591-600
Number of pages10
JournalBiochemistry
Volume53
Issue number3
DOIs
StatePublished - Jan 28 2014

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Amidohydrolases
Phosphoric Monoester Hydrolases
Nucleotides
Enzymes
Adenosine Monophosphate
Histidinol-Phosphatase
Catalytic Domain
Chromobacterium
Phosphoadenosine Phosphosulfate
Phosphates
Acyl Carrier Protein
Bacterial Genomes
Bacilli
Coenzyme A
Bacillus subtilis
Oligonucleotides
Superoxides
Adenosine
Escherichia coli
Sulfates

ASJC Scopus subject areas

  • Biochemistry

Cite this

Cummings, J. A., Vetting, M., Ghodge, S. V., Xu, C., Hillerich, B., Seidel, R. D., ... Raushel, F. M. (2014). Prospecting for unannotated enzymes: Discovery of a 3′,5′- nucleotide bisphosphate phosphatase within the amidohydrolase superfamily. Biochemistry, 53(3), 591-600. https://doi.org/10.1021/bi401640r

Prospecting for unannotated enzymes : Discovery of a 3′,5′- nucleotide bisphosphate phosphatase within the amidohydrolase superfamily. / Cummings, Jennifer A.; Vetting, Matthew; Ghodge, Swapnil V.; Xu, Chengfu; Hillerich, Brandan; Seidel, Ronald D.; Almo, Steven C.; Raushel, Frank M.

In: Biochemistry, Vol. 53, No. 3, 28.01.2014, p. 591-600.

Research output: Contribution to journalArticle

Cummings, Jennifer A. ; Vetting, Matthew ; Ghodge, Swapnil V. ; Xu, Chengfu ; Hillerich, Brandan ; Seidel, Ronald D. ; Almo, Steven C. ; Raushel, Frank M. / Prospecting for unannotated enzymes : Discovery of a 3′,5′- nucleotide bisphosphate phosphatase within the amidohydrolase superfamily. In: Biochemistry. 2014 ; Vol. 53, No. 3. pp. 591-600.
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