Crystal Structure of Thermostable p-nitrophenylphosphatase from Bacillus Stearothermophilus (Bs-TpNPPase)

Abstract

Thermostable p-nitrophenylphosphatase from Bacillus Stearothermophilus (Bs-TpNPPase) is involved in the Mg(2+)-dependent hydrolysis of the phosphoenzyme at an optimum reaction temperature of 55°C. Bs-TpNPPase has been cloned and overexpressed in the E.coli M15 strain. Based on the conserved active sites, the protein was suggested to be a member of the haloalkanoate dehalogenase (HAD) superfamily. Two site-specific point mutants of Bs-TpNPPase were prepared by changing the catalytic Asp10 and Thr43 to Ala10 and Ala43, respectively. The activity of the two mutants further confirms Bs-TpNPPase as a member of the HAD superfamily. HAD superfamily can be divided into the four subfamilies and play several biochemical roles such as DNA repair, signal transduction and secondary metabolism. To understand the relationship between structure and thermostability in HAD superfamily, Bs-TpNPPase from Bacillus Stearothermophilus was selected. The X-ray crystal structure of Bs-TpNPPase was determined at 1.5A resolution using the molecular replacement phasing method. The structure of Bs-TpNPPase has been deposited and the PDB code is 4KN8. Compared with Bsp, a mesophilic prokaryotic putative p-nitrophenyl phosphatase from Bacillus Subtilis, Bs- TpNPPase showed highly homology but variations in the level of leucine content, aromatic clusters, cation-Pi and hydrophobic interaction. These differences may affect the thermal stability of the protein. The crystal structure of Bs-TpNPPase described herein may serve as a guide to better understand the mechanism of thermostability and provide insights for further mutation work.

Bacillus subtilis 5′-nucleotidases with various functions and substrate specificities

“…The yutF gene encodes a protein that was predicted to be involved in N-acetyl-glucosamine catabolism and similar to 4-nitrophenyl phosphatase [24]. The condition-dependent transcriptome analysis revealed that yutF was almost constitutively expressed at low levels [25].…”

Structure of a His170Tyr mutant of thermostable pNPPase fromGeobacillus stearothermophilus

“…The reaction buffer contained 50 mM Tris-HCl pH 10.0, 1 mM MgCl 2 and 5 mM pNPP. Enzymes were incubated for 15 min at various temperatures ranging from 293 to 338 K. After the enzymes had been cooled on ice, their residual phosphatase activities were examined according to the method described by Guo et al (2013). Fitting of the collected data and calculation of T 1/2 were performed using GraphPad Prism 6.…”

“…The structure of wild-type TpNPPase (PDB entry 4kn8; Guo et al, 2013) was used as the search model. Refinement was carried out using PHENIX v.1.8.2-1309 (Terwilliger et al, 2009) and manual fitting of side chains was performed in Coot (Emsley & Cowtan, 2004).…”

“…An optimized conformational structure of proteins also plays an important role in enhancing thermostability, such as increased rigidity and decreased flexibility (Radestock & Gohlke, 2008, 2011, better packing (Fleming & Richards, 2000), more stable -helices and fewer cavities (Kumar et al, 2000;Li et al, 2005), deletion or shortening of loops (Russell et al, 1997) and oligomerization (Tanaka et al, 2004). In previous studies, X-ray diffraction of TpNPPase crystals was reported by Ji et al (2003) and the crystal structure was then determined by Guo et al (2013). Comparisons between the structures of TpNPPase and several thermolabile HADSF members were performed in order to demonstrate the possible favourable structural factors for protein thermostability.…”

“…Comparisons between the structures of TpNPPase and several thermolabile HADSF members were performed in order to demonstrate the possible favourable structural factors for protein thermostability. TpNPPase contains more leucines and additional aromatic pairs than its thermolabile homologues, and stabilizing the cap domain is also beneficial for enhancing the thermostability of HASDF enzymes (Guo et al, 2013).…”