| dc.description.abstract | Rapid advances in genome sequencing technology have created a wide gap between the number of available protein sequences, and reliable knowledge of their respective physiological functions. This work attempted to bridge this gap within the confines cog1387 and cog0613, from the polymerase and histidinol phosphatase (PHP) family of proteins, which is related to the amidohydrolase superfamily (AHS). The adopted approach involved using the mechanistic knowledge of a known enzymatic reaction, and discovering functions of closely related homologs using various tools including bioinformatics and rational library screening.
L-histidinol phosphate phosphatase (HPP) catalyzes the penultimate step in the biosynthesis of the amino acid: L-histidine. Recombinant HPP from L.lactis was purified and its metal content and activity were optimized. Mechanistic and structural studies were conducted using pH-Rate profiles, solvent isotope, viscosity effects, site-directed mutagenesis, and X-ray crystallography. These studies, along with extensive bioinformatic analysis, helped determine the boundaries of HPP activity among closely related enzyme sequences within cog1387.
Elen0235 from cog0613 was shown to hydrolyze 5-phosphoribose-1,2-cyclicphosphate (PRcP) to ribose-5-phosphate (R5P) and inorganic phosphate (Pi), with ribose-2,5-bisphosphate as a catalytic intermediate. The mechanism involved sequential hydrolysis of the two P-O bonds of the cyclic phosphodiester, by the attack of nucleophilic hydroxide at the P-center in both steps.
Cv1693 from cog0613 was shown to hydrolyze 3ʹ-phosphate from 3ʹ,5ʹ-diphosphoadenosine (pAp) and other diphosphonucleotides. However, enzymes from at least two other COGs, i.e. 1218 and 0618, are capable of hydrolyzing pAp. The authentic pAp-3ʹphosphatases from the three COGs under study were determined, and a thorough sequence network analysis of the respective COGs was performed. It was determined that the distribution of pAp-3ʹ-phosphatase from each of the 3 families is largely mutually exclusive in bacteria. This work supported the hypothesis that the physiological role of the target enzyme in vivo is the hydrolysis of pAp.
TrpH from Escherichia coli was found to hydrolyze pAp efficiently. However, when the substrate profile of this enzyme was probed further, this enzyme was found capable of hydrolyzing oligonucleotides bearing a 5ʹ-phosphoryl functional group with comparable efficiency. Further biochemical characterization revealed that this enzyme catalyzes the hydrolysis of oligoribonucleotides sequentially from 5ʹ→3ʹ. | en |
