Structure of HI0073 from Haemophilus influenzae, the nucleotide‐binding domain of a two‐protein nucleotidyl transferase

Introduction. Nucleotidyl transferases (NTs) are enzymes utilized by all living systems. They catalyze the transfer of a nucleotide to an acceptor hydroxyl group in a wide range of substrates. At the time of this writing, the International Union of Biochemistry and Molecular Biology classifies 61 NTs with distinct specificities [Enzyme Commission (EC) 2.7. 7, at http://www.chem.qmul.ac. uk/ iubmb/enzyme/], of which 3 have been reclassified as ribonucleases. NTs play roles in diverse biological processes such as polynucleotide synthesis and modification, small-molecule biosynthesis and metabolism, and antibiotic resistance. The 2 Haemophilus influenzae open reading frames, HI0073 and HI0074, comprise an operon coding for a novel 2-protein NT complex. We have shown by size exclusion chromatography (SEC) that HI0073 and HI0074 form a 2:2-heterotetramer in solution. HI0073 is a member of a protein sequence family from archaea and bacteria termed previously “minimal nucleotidyl transferases” (MNTs). We have also shown that HI0074 is structurally related to the substrate-binding domain of kanamycin nucleotidyl transferase. Taken together, these data support the premise that HI0073 is the nucleotide-binding domain, and HI0074 is the substrate-binding domain of the HI0073/74 enzyme complex. Moreover, sequence analysis revealed that the 2-component NTs form a superfamily comprised of at least 8 families, abundant in organisms living in harsh conditions and in some pathogens. The substrate specificity and the biological functions of the HI0073/74 family and of members of the other 2-component NT families are unknown. Here we present the crystal structure of the nucleotidebinding domain HI0073 at a resolution of 1.8 Å. The structure was determined by exploiting the anomalous signal of zinc. Intrinsic tryptophan fluorescence and the fluorescent compound 2 (or 3 )-O-(2,4,6-trinitrophenyl)adenosine 5 -triphosphate (TNP-ATP) were used to characterize nucleotide binding. Additional information about HI0073 and HI0074 is provided on our Structural Genomics website: http://s2f.umbi. umd.edu. Materials and Methods. Cloning of HI0073: The gene encoding HI0073 from H. influenzae Rd KW20 was amplified using PfuTurbo DNA polymerase (Stratagene; La Jolla, CA), genomic DNA, and 5 and 3 -end primers. For the forward primer, a sequence encoding a thrombin cleavage site and an NdeI restriction site at the 5 -end of the gene was included. The polymerase chain reaction (PCR) product was introduced into the pET100/D-TOPO expression vector by the TOPO directional cloning procedure (Invitrogen, La Jolla, CA). Recombinant plasmids were isolated from the Escherichia coli TOP10 strain. The expression construct for production of native protein without the His-tag was prepared by digestion with NdeI and self-ligation. Protein purification: HI0073 protein was purified from E. coli BL21(DE3) cells grown at 30°C in Luria–Bertani (LB) media containing 100 g/mL ampicillin to an A600 of 0.6 and induced with 0.1mM isopropylthio-D-galactoside (IPTG). Harvested cells were disrupted in buffer containing 20 mM Tris-HCl at pH 7.5 and 1 mM ethylenediaminetetraacetic acid (EDTA) (Buffer A) by passage through a French press. After centrifugation, the soluble fraction was passed through a Q Sepharose column (Amersham Biosciences) and eluted in buffer A using a 0–1 M NaCl linear gradient. Ammonium sulfate was added to a concentration of 2 M, and the solution was applied on a Butyl Sepharose column (Amersham Biosciences). The protein was eluted with a 2–0 M linear gradient of ammonium sulfate. The protein-containing fraction was applied on Sephacryl S-100 (Amersham Biosciences) equilibrated with 100 mM NaCl in Buffer A, and the eluted protein was concentrated to 15 mg/mL.