Crystallization and preliminary X-ray crystallographic analysis of PhoK, an extracellular alkaline phosphatase from Sphingomonas sp. BSAR-­1

A new alkaline phosphatase enzyme from Sphingomonas sp. strain BSAR-1, termed PhoK, has been shown to be useful in uranium bioprecipitation. PhoK has been expressed, purified and crystallized.

Abstract

Alkaline phosphatases (APs) are widely distributed from microbes to humans and are involved in several important biological processes such as phosphate nutrition, signal transduction and pathogenesis. Alkaline phosphatases are also useful in various industrial applications and in recombinant DNA technology. A new AP enzyme from Sphingomonas sp. strain BSAR-1, termed PhoK, has been shown to be useful in uranium bioprecipitation. PhoK was expressed, purified and crystallized. The crystals belonged to space group P4₃2₁2 or P4₁2₁2, with unit-cell parameters a = b = 87.37, c = 168.16 Å, and contained one enzyme molecule in the asymmetric unit. Native diffraction data have been collected to 1.95 Å resolution at the ESRF.

1. Introduction

Phosphates are essential for living organisms, which contain orthophosphates, pyrophosphates, polyphosphates, nucleotides, sugar phosphates and phosphorylated derivatives of organic compounds. Phosphatases play a crucial role in supporting microbial nutrition by releasing assimilable phosphate from various organic sources. The alkaline phosphatases constitute a superfamily of metalloenzymes that includes phosphatases, phosphodiesterases and sulfatases among others (Zalatan et al., 2006 ▶; Galperin et al., 1998 ▶). Alkaline phosphatases (EC 3.1.3.1), hereafter referred to as APs, are enzymes that hydrolyze phosphate monoesters to a phosphate ion and an alcohol at alkaline pH. The hydrolysis of phosphate monoesters is an important biological process that is involved in various metabolic and cellular signal-transduction pathways. APs are highly conserved from prokaryotes to eukaryotes (O’Brien & Herschlag, 2001 ▶).

The functions of APs are still not fully understood. In microbes they are involved in phosphorus nutrition, especially during phosphate starvation (Hou et al., 1966 ▶). Most APs are induced by free inorganic phosphate limitation, although constitutive AP activity has also been observed in bacteria located in the rumen (Forsberg & Cheng, 1980 ▶). Microbial APs also play indispensable roles in signal transduction (Hulett, 1996 ▶) and the virulence of bacteria (Kaduru­gamuwa & Beveridge, 1997 ▶). The availability of molecular sequence data has led to the identification of molecular families of phosphohydrolases for which signature sequence motifs have been defined (Thaller et al., 1998 ▶). A great divergence of enzyme characteristics has also been observed within these families (Berlutti et al., 2001 ▶; Goldman et al., 1990 ▶; Gomez & Ingram, 1995 ▶; Wagner et al., 1995 ▶).

The catalytic reaction of APs with various synthetic chromogenic substrates has led to their usage in immunodetection techniques such as ELISA and Western blotting (Tomazic-Allen, 1991 ▶). They are also used in recombinant DNA technology. We recently reported an AP, termed PhoK, from Sphingomonas sp. strain BSAR-1 (Nilgiriwala et al., 2008 ▶). PhoK was found to exhibit several unique features, such as constitutive expression, extracellular release, a very large molecular size of the active protein (∼200 kDa) and high specific activity. We also reported the successful application of a recombinant Escherichia coli strain overexpressing PhoK for the bioprecipitation of uranium as uranyl phosphate under alkaline conditions (Nilgiriwala et al., 2008 ▶). Considering the possible potential of PhoK in various applications, it was considered desirable to understand the structure–function relationships of this protein. With this aim, the crystallization of PhoK was initiated. In this communication, we report the crystallization and preliminary X-ray crystallographic analysis of purified recombinant PhoK enzyme from Sphingomonas sp. strain BSAR-1.

2. Materials and methods

2.1. Overexpression and purification of PhoK protein

The cloning of the phoK gene from Sphingomonas sp. BSAR-1 in E. coli BL21 (DE3) pLysS has been reported previously (Nilgiriwala et al., 2008 ▶). The PhoK-overexpressing recombinant E. coli strain, denoted EK4, was grown aerobically at 310 K in Luria–Bertani (LB) medium containing 33 µg ml⁻¹ chloramphenicol and 50 µg ml⁻¹ kanamycin until the culture attained an OD_600nm of 0.8. The cells were subsequently induced by 1 mM isopropyl β-d-1-thiogalacto­pyranoside (IPTG) at 303 K for 4 h under agitation at 180 rev min⁻¹. The recombinant (His₆-tagged) PhoK-overexpressing cells were lysed by sonication (Branson, Germany) in 100 mM Tris, 100 mM NaCl buffer containing 0.5 M urea. The enzyme was purified from the soluble cell-free lysate by Ni²⁺–nitrilotriacetic acid (Ni²⁺–NTA) affinity chromatography. All purification steps were carried out at 277 K and as per QIAexpressionist protocols (Qiagen, USA).

The purified recombinant protein was dialyzed against 50 mM Tris, 100 mM NaCl buffer at 277 K. The protein was then concentrated using a Centricon centrifugal concentrator (10 000 Da molecular-mass cutoff). The purity of the protein was determined after resolving it by SDS–PAGE on 10% resolving gel with Coomassie Brilliant Blue staining. The concentrated PhoK protein was used for crystallization without removing the His tag.

2.2. Crystallization of PhoK

Screening for crystallization conditions was performed by the sitting-drop vapour-diffusion method in 96-well crystallization plates (Greiner) using a CyBio HTPC robot operated at 298 K. Drops were prepared by mixing protein solution (3–5 mg ml⁻¹) with an equal volume of reservoir solution and were equilibrated against 75 µl reservoir solution. Initial screening was performed using commercially available crystallization screens (Structure Screens I and II from Molecular Dimensions Ltd and Wizard I and II from Emerald BioSystems).

2.3. X-ray diffraction data collection

For X-ray diffraction data collection, crystals were soaked in mother liquor containing 25% glycerol for 10–20 s and flash-cooled in liquid nitrogen. X-ray diffraction data were collected at 100 K on the FIP-BM30A beamline at the ESRF (Roth et al., 2002 ▶) using the oscillation method. A total of 110 frames were collected at a crystal-to-detector distance of 269.10 mm with 1.0° oscillation per frame and an exposure time of 30 s. The diffraction data were processed using the XDS software suite (Kabsch, 1993 ▶).

3. Results and discussion

Crystals of the purified protein appeared under several conditions from the crystallization screens. Crystals grown in these screens were used directly for diffraction studies. The present analysis was con­ducted using a crystal grown in the condition 1.6 M ammonium sulfate, 10%(v/v) dioxane, 100 mM MES pH 6.5 (Fig. 1 ▶). The crystal used for diffraction data collection was approximately 0.1 × 0.07 × 0.07 mm in size. The crystal belonged to space group P4₃2₁2 or P4₁2₁2, with unit-cell parameters a = b = 87.37, c = 168.16 Å. The calculated V _M value (Matthews, 1968 ▶) of 2.55 ų Da⁻¹ suggested the presence of one protein molecule per asymmetric unit, with a solvent content of 51.82%. The crystal diffracted to 1.95 Å resolution at 100 K. Preparation of a selenomethionine derivative is in progress. Structure solution will be attempted using the molecular-replacement and MAD/SAD methods.

Figure 1.

Single crystal of PhoK from Sphingomonas sp. BSAR-1 in a crystallization drop of total volume 1.8 µl.

Table 1. Data-collection and processing statistics.

Values in parentheses are for the highest resolution shell.

X-ray source	FIP-BM30A beamline, ESRF
Wavelength (Å)	0.976180
Temperature (K)	100
Resolution range (Å)	50–1.95 (2.05–1.95)
Space group	P43212 or P41212
Unit-cell parameters (Å)	a = 87.37, b = 87.37, c = 168.16
No. of reflections measured	365046
No. of unique reflections	82621
No. of molecules in ASU	1
Completeness (%)	91.1 (78.6)
I/σ(I)	13.75 (2.61)
Rmrgd-F† (%)	9.9 (52.7)
Mosaicity (°)	0.155

^†Diederichs & Karplus (1997 ▶).