Prospecting for Unannotated Enzymes: Discovery of a 3鈥?5鈥?Nucleotide Bisphosphate Phosphatase within the Amidohydrolase Superfamily

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• 作者：Jennifer A. Cummings ; Matthew Vetting ; Swapnil V. Ghodge ; Chengfu Xu ; Brandan Hillerich ; Ronald D. Seidel ; Steven C. Almo ; Frank M. Raushel
• 刊名：Biochemistry
• 出版年：2014
• 出版时间：January 28, 2014
• 年：2014
• 卷：53
• 期：3
• 页码：591-600
• 全文大小：638K
• ISSN：1520-4995

文摘

In bacteria, 3鈥?5鈥?adenosine bisphosphate (pAp) is generated from 3鈥?phosphoadenosine 5鈥?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鈥?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 脜 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 k_cat and k_cat/K_m for the hydrolysis of pAp are 22 s^鈥? and 1.4 脳 10⁶ M^鈥? s^鈥?, respectively. The enzyme is promiscuous and is able to hydrolyze other 3鈥?5鈥?bisphosphonucleotides (pGp, pCp, pUp, and pIp) and 2鈥?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.