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Title: Crystallization of human nicotinamide phosphoribosyltransferase

Abstract

Human nicotinamide phosphoribosyltransferase has been crystallized using microseeding methods and X-ray diffraction data have been collected at 2.0 Å resolution. In the NAD biosynthetic pathway, nicotinamide phosphoribosyltransferase (NMPRTase; EC 2.4.2.12) plays an important role in catalyzing the synthesis of nicotinamide mononucleotide from nicotinamide and 5′-phosphoribosyl-1′-pyrophosphate. Because the diffraction pattern of the initally obtained crystals was not suitable for structure analysis, the crystal quality was improved by successive use of the microseeding technique. The resultant crystals diffracted to 2.0 Å resolution. These crystals belonged to space group P21, with unit-cell parameters a = 60.56, b = 106.40, c = 82.78 Å. Here, the crystallization of human NMPRTase is reported in the free form; the crystals should be useful for inhibitor-soaking experiments on the enzyme.

Authors:
; ;  [1];  [1];  [2];  [1]
  1. Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan)
  2. (Japan)
Publication Date:
OSTI Identifier:
22360327
Resource Type:
Journal Article
Resource Relation:
Journal Name: Acta Crystallographica. Section F; Journal Volume: 63; Journal Issue: Pt 5; Other Information: PMCID: PMC2335003; PMID: 17565174; PUBLISHER-ID: gj5014; OAI: oai:pubmedcentral.nih.gov:2335003; Copyright (c) International Union of Crystallography 2007; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United Kingdom
Language:
English
Subject:
75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND SUPERFLUIDITY; CRYSTALLIZATION; CRYSTALS; RESOLUTION; SPACE GROUPS; SYNTHESIS; X-RAY DIFFRACTION

Citation Formats

Takahashi, Ryo, Nakamura, Shota, Yoshida, Takuya, Kobayashi, Yuji, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, and Ohkubo, Tadayasu, E-mail: ohkubo@phs.osaka-u.ac.jp. Crystallization of human nicotinamide phosphoribosyltransferase. United Kingdom: N. p., 2007. Web. doi:10.1107/S1744309107006069.
Takahashi, Ryo, Nakamura, Shota, Yoshida, Takuya, Kobayashi, Yuji, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, & Ohkubo, Tadayasu, E-mail: ohkubo@phs.osaka-u.ac.jp. Crystallization of human nicotinamide phosphoribosyltransferase. United Kingdom. doi:10.1107/S1744309107006069.
Takahashi, Ryo, Nakamura, Shota, Yoshida, Takuya, Kobayashi, Yuji, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, and Ohkubo, Tadayasu, E-mail: ohkubo@phs.osaka-u.ac.jp. Tue . "Crystallization of human nicotinamide phosphoribosyltransferase". United Kingdom. doi:10.1107/S1744309107006069.
@article{osti_22360327,
title = {Crystallization of human nicotinamide phosphoribosyltransferase},
author = {Takahashi, Ryo and Nakamura, Shota and Yoshida, Takuya and Kobayashi, Yuji and Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094 and Ohkubo, Tadayasu, E-mail: ohkubo@phs.osaka-u.ac.jp},
abstractNote = {Human nicotinamide phosphoribosyltransferase has been crystallized using microseeding methods and X-ray diffraction data have been collected at 2.0 Å resolution. In the NAD biosynthetic pathway, nicotinamide phosphoribosyltransferase (NMPRTase; EC 2.4.2.12) plays an important role in catalyzing the synthesis of nicotinamide mononucleotide from nicotinamide and 5′-phosphoribosyl-1′-pyrophosphate. Because the diffraction pattern of the initally obtained crystals was not suitable for structure analysis, the crystal quality was improved by successive use of the microseeding technique. The resultant crystals diffracted to 2.0 Å resolution. These crystals belonged to space group P21, with unit-cell parameters a = 60.56, b = 106.40, c = 82.78 Å. Here, the crystallization of human NMPRTase is reported in the free form; the crystals should be useful for inhibitor-soaking experiments on the enzyme.},
doi = {10.1107/S1744309107006069},
journal = {Acta Crystallographica. Section F},
number = Pt 5,
volume = 63,
place = {United Kingdom},
year = {Tue May 01 00:00:00 EDT 2007},
month = {Tue May 01 00:00:00 EDT 2007}
}
  • Herein we disclose SAR studies that led to a series of isoindoline ureas which we recently reported were first-in-class, non-substrate nicotinamide phosphoribosyltransferase (NAMPT) inhibitors. Modification of the isoindoline and/or the terminal functionality of screening hit 5 provided inhibitors such as 52 and 58 with nanomolar antiproliferative activity and preclinical pharmacokinetics properties which enabled potent antitumor activity when dosed orally in mouse xenograft models. X-ray crystal structures of two inhibitors bound in the NAMPT active-site are discussed.
  • Purpose: Radiation therapy for head and neck cancer commonly leads to radiation sialadenitis. Emerging evidence has indicated that phenylephrine pretreatment reduces radiosensitivity in the salivary gland; however, the underlying cytoprotective mechanism remains unclear. Nicotinamide phosphoribosyltransferase (NAMPT) is not only a key enzyme for the nicotinamide adenine dinucleotide salvage pathway, but also a cytokine participating in cell survival, metabolism, and longevity, with a broad effect on cellular functions in physiology and pathology. However, the regulatory events of NAMPT in response to the irradiated salivary gland are unknown. Methods and Materials: The cell viability of primary cultured submandibular gland cells was determinedmore » using the PrestoBlue assay. NAMPT expression was measured using reverse transcriptase polymerase chain reaction and Western blotting in vitro and in vivo. Silent information regulator 1 (SIRT1) and phosphorylated Akt protein levels were examined by Western blotting. The cellular locations of NAMPT and SIRT1 were detected by immunohistochemistry. NAMPT promoter activity was assessed using the luciferase reporter gene assay. Results: NAMPT was mainly distributed in the cytoplasm of granular convoluted tubule cells and ductal cells in normal submandibular glands. mRNA and protein expression of NAMPT was downregulated after radiation but upregulated with phenylephrine pretreatment both in vivo and in vitro. Moreover, the protein expression of phosphorylated Akt and SIRT1 was decreased in irradiated glands, and phenylephrine pretreatment restored the expression of both. SIRT1 was mainly located in the cell nucleus and cytoplasm in the normal submandibular gland. Phenylephrine dramatically enhanced the expression of SIRT1, which was significantly reduced by radiation. Furthermore, phenylephrine induced a marked increase of NAMPT promoter activity. Conclusions: These findings reveal the regulatory mechanisms of NAMPT expression, which help to understand the mechanism of the cytoprotective role of phenylephrine on irradiated tissues.« less
  • The virtually complete deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) results in a devastating neurological disease, Lesch-Nyhan syndrome. Transfer of the HPRT gene into fibroblasts and lymphoblasts in vitro and into hematopoietic cells in vivo has been accomplished by other groups with retroviral-derived vectors. It appears to be necessary, however, to transfer the HPRT gene into neuronal cells to correct the neurological dysfunction of this disorder. The neurotropic virus herpes simplex virus type 1 has features that make it suitable for use as a vector to transfer the HPRT gene into neuronal tissue. This report describes the isolationmore » of an HPRT-deficient rat neuroma cell line, designated B103-4C, and the construction of a recombinant herpes simplex virus type 1 that contained human HPRT cDNA. These recombinant viruses were used to infect B103-4C cells. Infected cells expressed HPRT activity which was human in origin.« less
  • This research regarding the differential expression of the hypoxanthine phosphoribosyltransferase (HPRT) gene concludes that an element of the gene interacts with different nuclear proteins. Binding sites for nuclear proteins from both neuronal and nonneuronal sources were mapped. 62 refs., 9 figs.