Strucutural and biochemical characterization of E. coli HinT and its H101A mutant

• Published in: The FASEB journal 2007-04, Vol.21 (5), p.A276-A276
• Main Authors: Cody, Vivian, Pace, Jim, Snell, Edward, Chou, Tsui‐fen, Wagner, Conrad R.
• Format: Article
• Language: English
• ISSN: 0892-6638; 1530-6860
• DOI: 10.1096/fasebj.21.5.A276

The histidine triad nucleotide proteins (Hints) from human and E. coli have been established as purine nucleoside phosphoramidases. Human Hint1 possesses tumor suppressor activity whereas E. coli HinT is involved in high salt tolerance for E. coli. The homodimeric Hint proteins are characterized by a His‐X‐His‐X‐His‐XX motif where X is a hydrophobic residue. Additionally, human HinT1 and its bacterial homolog have different substrate specificities. To understand the mechanism of substrate specificity, kinetic and structural studies of mutants that replace the active site histidines were carried out. These data showed that H101A decreased specific activity by 105‐fold. Diffraction data were measured for the wild type (1.5Å resolution) and H101A mutant (1.7 Ǻ) ecHinT GMP complexes. There are four unique homodimers in the wild type, and two homodimers in the mutant structure. The overall fold of the ecHinT structures is similar to human HinT1. The current refinement model for both structures reveals that although the region encompassing the His triad region is similar to the human HinT1 complex, there are larger shifts in the alignment of the backbone and conformational changes in a number of other regions. In the H101A mutant, loss of the hydrogen bond interaction between His101 and the phosphate oxygen of GMP is critical for the catalytic role of H101. Supported in part by University of Minnesota AHC‐FRD grant (CRW).