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Lee, Jae Young; Kwak, Jae; Moon, Jinho; Eom, Soo Hyun; Liong, E.C.; Pédelacq, J.D.; Berendzen, Joel; Suh, Se Won

doi:10.1038/nsb0901-789

Cited by 211 publications

(68 citation statements)

References 26 publications

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“…Our biochemical studies of the E. coli SurE and the previous data on two SurE proteins from the thermophilic bacterium Thermotoga maritima (17,38) and from the archaebacterium Pyrobaculum aerophilum (39) clearly demonstrated that the annotation of SurE proteins as acid phosphatases is not accurate. Acid phosphatases (EC 3.1.3.2) comprise a large group of nonspecific phosphohydrolases capable to hydrolyze a broad range of phosphorylated sugars, amino acids, nucleoside mono-, di-, and triphosphates (BRENDA data base, www.brenda.uni-koeln.de/).…”

Section: Screening Of Purified Proteins For Phosphatase Activity-mentioning

confidence: 94%

“…In HAD-like phosphohydrolases, the metal cofactor (usually Mg 2ϩ ) is involved in the coordination of the nucleophilic side-chain carboxylate of catalytic Asp and the phosphoryl group of the substrate (36,37). Recent structural studies of SurE proteins from T. maritima and P. aerophilum revealed the presence in the active site of mononuclear metal center chelated by several conserved residues, including the strictly conserved N-terminal DD pair (17,38,39). The threedimensional structure of the N-terminal fragment of the bifunctional Rel/Spo homologue (the HD domain protein) from Streptococcus dysgalactiae identified several conserved residues (His-53, His-77, Asp-78, and Asp-144) involved in the coordination of Mn 2ϩ (40).…”

Section: Screening Of Purified Proteins For Phosphatase Activity-mentioning

confidence: 99%

See 1 more Smart Citation

Interaction of Genome-linked Protein (VPg) of Turnip Mosaic Virus with Wheat Germ Translation Initiation Factors eIFiso4E and eIFiso4F

Khan

Mizumoto

Ray

et al. 2006

Journal of Biological Chemistry

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To find proteins with nucleotidase activity in Escherichia coli, purified unknown proteins were screened for the presence of phosphatase activity using the general phosphatase substrate p-nitrophenyl phosphate. Proteins exhibiting catalytic activity were then assayed for nucleotidase activity against various nucleotides. ). Further biochemical characterization of SurE revealed that it has a broad substrate specificity and can dephosphorylate various ribo-and deoxyribonucleoside 5-monophosphates and ribonucleoside 3-monophosphates with highest affinity to 3-AMP. SurE also hydrolyzed polyphosphate (exopolyphosphatase activity) with the preference for short-chain-length substrates (P 20 -25 ). YfbR was strictly specific to deoxyribonucleoside 5-monophosphates, whereas YjjG showed narrow specificity to 5-dTMP, 5-dUMP, and 5-UMP. The three enzymes also exhibited different sensitivities to inhibition by various nucleoside di-and triphosphates: YfbR was equally sensitive to both di-and triphosphates, SurE was inhibited only by triphosphates, and YjjG was insensitive to these effectors. The differences in their sensitivities to nucleotides and their varied substrate specificities suggest that these enzymes play unique functions in the intracellular nucleotide metabolism in E. coli.DNA and RNA synthesis requires a continuous and balanced supply of the four deoxyribonucleotides and four ribonucleotides. A network of biosynthetic and catabolic enzymes regulates the size of each pool with the enzymes ribonucleotide reductase, nucleoside kinases, and nucleotidases playing the main roles (1). By opposing the phosphorylation of nucleosides by kinases, intracellular 5Ј-nucleotidases participate in substrate cycles that regulate the cellular levels of ribo-and deoxyribonucleoside monophosphates and thereby all ribo-and deoxyribonucleotide pools (1, 2).Nucleoside monophosphate phosphohydrolases or nucleotidases (EC 3.1.3.5 and EC 3.1.3.6) are phosphatases that specifically dephosphorylate nucleoside monophosphates to nucleosides and inorganic phosphate. Seven mammalian 5Ј-nucleotidases have been identified through cloning and biochemical characterization. These enzymes differ in tissue specificity, subcellular localization, primary structure, and substrate specificity (2). All act on nucleoside monophosphates producing free nucleosides and P i . Each natural nucleotide can be the substrate for several nucleotidases, because they have overlapping specificities. The ubiquitous ecto-5Ј-nucleotidase, eN, 1 is anchored to the surface of the plasma membrane, and AMP is considered to be its major physiological substrate (3). Five other nucleotidases, including dNT-1, occur in the cytosol and one (dNT-2) occurs in mitochondria. dNT-1 and dNT-2 show a preference for the dephosphorylation of dUMP and dTMP (4). The "high K m -nucleotidase" cN-II prefers GMP and IMP (5), the cytosolic nucleotidases cN-IA and cN-IB have a preference for dephosphorylation of AMP (6, 7), and PN-1 (or cN-III) is most active with CMP and UMP (8). For bovine cN-II...

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Section: Screening Of Purified Proteins For Phosphatase Activity-mentioning

confidence: 94%

Section: Screening Of Purified Proteins For Phosphatase Activity-mentioning

confidence: 99%

Interaction of Genome-linked Protein (VPg) of Turnip Mosaic Virus with Wheat Germ Translation Initiation Factors eIFiso4E and eIFiso4F

Khan

Mizumoto

Ray

et al. 2006

Journal of Biological Chemistry

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“…As the three-dimensional structure of proteins is intimately coupled with the molecular function, the structure of a protein may provide clues for its molecular function. The validity of this approach has been demonstrated by several examples (1)(2)(3), and a number of large-scale structural genomics projects have been initiated (4,5).…”

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confidence: 99%

Directed evolution approach to a structural genomics project: Rv2002 from Mycobacterium tuberculosis

Yang

Park

Waldo

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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One of the serious bottlenecks in structural genomics projects is overexpression of the target proteins in soluble form. We have applied the directed evolution technique and prepared soluble mutants of the Mycobacterium tuberculosis Rv2002 gene product, the wild type of which had been expressed as inclusion bodies in Escherichia coli. A triple mutant I6T͞V47M͞T69K (Rv2002-M3) was chosen for structural and functional characterizations. Enzymatic assays indicate that the Rv2002-M3 protein has a high catalytic activity as a NADH-dependent 3␣, 20␤-hydroxysteroid dehydrogenase. We have determined the crystal structures of a binary complex with NAD ؉ and a ternary complex with androsterone and NADH. The structure reveals that Asp-38 determines the cofactor specificity. The catalytic site includes the triad Ser-140͞Tyr-153͞ Lys-157. Additionally, it has an unusual feature, Glu-142. Enzymatic assays of the E142A mutant of Rv2002-M3 indicate that Glu-142 reverses the effect of Lys-157 in influencing the pKa of Tyr-153. This study suggests that the Rv2002 gene product is a unique member of the SDR family and is likely to be involved in steroid metabolism in M. tuberculosis. Our work demonstrates the power of the directed evolution technique as a general way of overcoming the difficulties in overexpressing the target proteins in soluble form.

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“…The predictions can provide a basis for assigning a molecular function to the hypothetical protein. For instance, Zarembinski et al (1998), Hwang et al (1999) and Lee et al (2001) have succeeded in structure-based assignment of the molecular function of hypothetical proteins. The overall conformation, detection of the active site and the amino-acid composition of the active site, which can be revealed from the structure of YedU, would lead us to the elucidation of its biochemical function.…”

Section: Introductionmentioning

confidence: 99%

Crystallization and preliminary X-ray crystallographic analysis of ayedUgene product fromEscherichia coli

Kim

et al. 2002

Acta Crystallogr D Biol Cryst

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A yedU gene product with a molecular mass of 31 kDa is a hypothetical protein with no known function. The protein was puri®ed and crystallized at 296 K. X-ray diffraction data have been collected to 2.3 A Ê using synchrotron radiation. The crystals belong to the primitive orthorhombic system, with unit-cell parameters a = 50.56, b = 63.45, c = 168.02 A Ê . The asymmetric unit contains two monomers of the protein, with a corresponding V M of 2.25 A Ê 3 Da À1 and a solvent content of 44.84%.

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Untitled

Cited by 211 publications

References 26 publications

Interaction of Genome-linked Protein (VPg) of Turnip Mosaic Virus with Wheat Germ Translation Initiation Factors eIFiso4E and eIFiso4F

Interaction of Genome-linked Protein (VPg) of Turnip Mosaic Virus with Wheat Germ Translation Initiation Factors eIFiso4E and eIFiso4F

Directed evolution approach to a structural genomics project: Rv2002 from Mycobacterium tuberculosis

Crystallization and preliminary X-ray crystallographic analysis of ayedUgene product fromEscherichia coli

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