AraL sugar phosphatase from B. subtilis L. M. Godinho and I. de Sa´ -Nogueira motif I ------------------------------MRIMASHDTPVSPAGILIDLDGTVFRGNEL 30 ----------------------------------------MTIKNVICDIDGVLMHDNVA 20 -------------------MTAQQGVPIKITNKEIAQEFLDKYDTFLFDCDGVLWLGSQA 41 -------------------------MAKKLSSPKEYKEFIDKFDVFLFDCDGVLWSGSKP 35 MALIYSSDKKDDDIINVEKKYESFLKEWNLNKMINSKDLCLEFDVFFFDCDGVLWHGNEL 60 .: * **.: .. ARAL_BACSU NAGD_ECOLI PNPP_YEAST PNPP_SCHPO A5PGW7_PLAFA motif II IEGAREAIKTLRRMGKKIVFLSNRGNISRAMCRKKLLGAGIE-TDVNDIVLSSSVTAAFL 89 ARAL_BACSU VPGAAEFLHGIMDKGLPLVLLTNYPSQTGQDLANRFATAGVD-VPDSVFYTSAMATADFL 79 NAGD_ECOLI LPYTLEILNLLKQLGKQLIFVTNNSTKSRLAYTKKFASFGID-VKEEQIFTSGYASAVYI 100 PNPP_YEAST IPGVTDTMKLLRSLGKQIIFVSNNSTKSRETYMNKINEHGIA-AKLEEIYPSAYSSATYV 94 PNPP_SCHPO IEGSIEVINYLLREGKKVYFITNNSTKSRASFLEKFHKLGFTNVKREHIICTAYAVTKYL 120 A5PGW7_PLAFA : : :: : * : :::* . : ::: *. . . : :. : :: KKHYRF----SKVWVLGEQGLVDELRLAGVQNASEP---------------------KEA 124 RRQEGK-----KAYVVGEGALIHELYKAGFTITDVN-----------------------P 111 RDFLKLQPGKDKVWVFGESGIGEELKLMGYESLGGADSRLDTPFDAAKSPFLVNGLDKDV 160 KKVLKL-PADKKVFVLGEAGIEDELDRVGVAHIGGTDPSLRR--ALASEDVEKIGPDPSV 151 YDKEEYRLRKKKIYVIGEKGICDELDASNLDWLGGSNDNDKK---IILKDDLGIIVDKNI 177 * :*.** .: .** . . cap domain DWLVISLHETLTYDDLNQAFQAAAG-GARIIATNKDRSFPNEDGNAIDVAGMIGAIETSA 183 DFVIVGETRSYNWDMMHKAAYFVAN-GARFIATNPDTH----GRGFYPACGALCAGIEKI 166 SCVIAGLDTKVNYHRLAVTLQYLQKDSVHFVGTNVDST-FPQKGYTFPGAGSMIESLAFS 219 GAVLCGMDMHVTYLKYCMAFQYLQDPNCAFLLTNQDST-FPTNGKFLPGSGAISYPLIFS 210 GAVVVGIDFNINYYKIQYAQLCINELNAEFIATNKDATGNFTSKQKWAGTGAIVSSIEAV 237 . :: . .: : . :: ** * * : motif III motif IV QAKTELVVGKPSWLMAEAACTAMGLSAHECMIIGDSIESDIAMGKLYGMK-SALVLTGSA 242 SGRKPFYVGKPSPWIIRAALNKMQAHSEETVIVGDNLRTDILAGFQAGLE-TILVLSGVS 225 SNRRPSYCGKPNQNMLNSIISAFNLDRSKCCMVGDRLNTDMKFGVEGGLGGTLLVLSGIE 279 TGRQPKILGKPYDEMMEAIIANVNFDRKKACFVGDRLNTDIQFAKNSNLGGSLLVLTGVS 270 SLKKPIVVGKPNVYMIENVLKDLNIHHSKVVMIGDRLETDIHFAKNCNIK-SILVSTGVT 296 : *** : . . : ::** :.:*: . .: : ** :* KQG-----EQRLYTPDYVLDSIKDVTKLAEEGILI 272 SLD---DIDSMPFRPSWIYPSVAEIDVI------- 250 TEERALKISHDYPRPKFYIDKLGDIYTLTNNEL-- 312 KEEEILEKDAP-VVPDYYVESLAKLAETA------ 298 NANIYLNHNSLNIHPDYFMKSISELL--------- 322 . . *.: .: .: ARAL_BACSU NAGD_ECOLI PNPP_YEAST PNPP_SCHPO A5PGW7_PLAFA ARAL_BACSU NAGD_ECOLI PNPP_YEAST PNPP_SCHPO A5PGW7_PLAFA ARAL_BACSU NAGD_ECOLI PNPP_YEAST PNPP_SCHPO A5PGW7_PLAFA ARAL_BACSU NAGD_ECOLI PNPP_YEAST PNPP_SCHPO A5PGW7_PLAFA Fig. 2. Alignment of AraL with other pNPP- ases members of the HAD superfamily (sub- family IIA). The amino acid sequences of AraL from B. subtilis (P94526), NagD from E. coli (P0AF24), the pNPPases from S. cerevisiae (P19881), Sz. pombe (Q00472) and P. falciparum (A5PGW7) were aligned using CLUSTAL W2 [41]. Similar (‘.’ and ‘:’) and identical (‘*’) amino acids are indicated. Gaps in the amino acid sequences inserted to optimize alignment are indicated by a dash (–). The motifs I, II, III and IV of the HAD superfamily and the cap domain C2 are boxed. Open arrowheads point to the catalytic residues in motifs I–IV. Identical residues in all ﬁve sequences, and identical residues in at least three sequences, are highlighted in dark and light grey, respectively. of AraL in pET30a, with a small deletion at the N-ter- activity at pH 7 (Fig. 4). Although, at pH 8 and 9, the minus (pLG11; Fig. 1). Production of this truncated activity was considerably lower than that observed at version of AraL was achieved in E. coli BL21 pLys(S) pH 7, the values are higher than that observed at pH 6, DE3 cells harboring pLG12, after IPTG induction, and no activity was measured below pH 4. The optimal although the protein was obtained in the insoluble frac- temperature was analyzed over temperatures in the tion (data not shown). Thus, overproduction was range 25–70 C. The enzyme was most active at 65 C attempted using the auto-induction method described and, at 25 C, no activity was detected (Fig. 4). These by Studier [22]. In the soluble and insoluble fractions of biophysical AraL properties fall into the range found cells harboring pLG11, a protein of 29 kDa was for other characterized phosphatases from B. subtilis: detected, which matched the predicted size for the pH 7–10.5 and 55–65 C [23–27]. recombinant AraL (Fig. 3A). The protein was puriﬁed to more than 95% homogeneity by Ni2+-nitrilotriacetic acid agarose afﬁnity chromatography (Fig. 3B). Characterization of AraL AraL phosphatase activity was measured using the syn- HAD superfamily proteins typically employ a biva- lent metal cation in catalysis, and phosphatases, partic- ularly those belonging to the subclass IIA, frequently use Mg2+ as a cofactor [3,6,8,13]. The effect of diva- lent ions (Mg2+, Zn2+, Mn2+, Ni2+, Co2+) in AraL activity was tested and the results obtained indicated that catalysis absolutely requires the presence of Mg2+ thetic substrate 4-nitrophenyl phosphate (pNPP). AraL (Fig. 4). The addition of EDTA to a reaction contain- is characterized as a neutral phosphatase with optimal ing MgCl2, prevented AraL activity (data not shown). 2514 FEBS Journal 278 (2011) 2511–2524 ª 2011 The Authors Journal compilation ª 2011 FEBS