Supplemental information for“A New Class of Adenylate Kinase in Methanogens is related to Uridylate Kinase”

Laura L. Grochowski, Kaitlin Censky, Huimin Xu, and Robert H. White*.

Methods

Chemicals. ATP, AMP, ADP, GTP, GMP, UMP, UTP, and all other chemicals were obtained from Sigma.

Cloning and Overexpression of the M. jannaschii MJ0458 Gene in E. coli. The MJ0458 gene (Swiss-Prot accession number Q57900) was amplified by PCR from

genomic DNA using oligonucleotide primers MJ0458 –Fwd (5’- GGTGGTCATATGCATATAGTAAAAATTGG-3’) and MJ0458-Rev (5’- GATCGGATCCTTATATTTTATCTATTCC-3’). PCR amplification was performed as described previously (1) using a 55 C annealing temperature. Purified PCR product was digested with Nde 1 and Bam H1restriction enzymes and ligated into compatible sites in plasmid pT7-7. Sequence of the resulting plasmid, pMJ0458, was verified by DNA sequencing. pMJ0458 was transformed into E. coli strain BL21 (DE3)-RIL.Transformed cells were grown in LB medium (200 mL) supplemented with 100 g/mL ampicillin at 37 C with shaking until they reached an OD600 of 1.0. Recombinant protein production was induced by addition of lactose to a final concentration of 28 mM. After an additional 4 hours of culture at 37 C, the cells were harvested by centrifugation (4,000 g, 5 min) and frozen at –20 C. Induction of the desired protein was confirmed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of total cellular proteins.

Purification of Recombinant AdkB. E. coli cells expressing MJ0458 were resuspended in 4 mL extraction buffer (50 mM N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES) pH 7.0, 10 mM MgCl2, 20 mM DTT) and lysed by sonication. After precipitating the majority of E. coli proteins by heating the cell lysate in an 80 C waterbath for 10 min, the MJ0458-derived protein was purified by anion exchange chromatography on a MonoQ HR column (1 x 8 cm, Amersham Bioscience) with a linear gradient from 0-1 M NaCl in 25 mM Tris pH 7.5 over 55 mL at 1 mL/min. A single protein band, corresponding to the expected MJ0458 molecular mass of 24 kDa, was observed in fractions eluting around 230 mM NaCl. Protein concentration was determined by Bradford analysis (2). The identity of the purified enzyme was confirmed by MALDI mass spectral analysis of the excised protein band from the polyacrylamide gel, following in-gel trypsin digestion, using a 4800 MALDI Tof/Tof mass spectrometer (Applied Biosystems).

The native molecular weight of MJ0458 was determined by size exclusion chromatography on a Superose 12HR column (10 x 300 mm) with a buffer containing 50 mM TES pH 7.5, 300 mM NaCl at 0.5 mL/min with detection at 280 nm. Protein standards used to generate the standard curve included apoferritin (443 kDa), β-amylase (200 kDa), conalbumin (77 kDa); bovine serum albumin (66 kDa), carbonic anhydrase (29 kDa), cytochrome C (14 kDa) and B12 (1.4 kDa).

Adenylate Kinase Activity Assay. Adenylate kinase activity was determined by measuring the generation of ADP through a coupled assay with pyruvate kinase and lactate dehydrogenase (3). The standard assay was a continuous assay at 37 C and included 3.5 units pyruvate kinase, 5 units lactate dehydrogenase,25 mM TES/K+ buffer pH 7.0, 5 mM MgCl2, 10 mM DTT, 1mM ATP, 1 mM AMP, 2 mM phosphoenol pyruvate, 0.5 mM NADH in a final volume of 1 ml. Following equilibration of the above mixture to the desired temperature (10 min), the reaction was initiated with the addition of AdkB (1 g) and an initial linear decrease in absorbance at 340 nm was observed over 5 minutes. Although ADP is the preferred nucleotide substrate for pyruvate kinase, it also utilizes a number of alternate nucleotides including GDP (4, 5) thus allowing us to determine if AdkB could utilize alternate nucleotidyl donors or acceptors. Assays for alterate phosphoryl donors and acceptors were conducted under the standard conditions with 1 mM each donor and acceptor substrate.Succinate, L-aspartate, L-glutamate, diaminopimelate were all tested for phosphorylation by AdkBwith ATP at concentrations up to 20 mM. For kinetic assays AMP concentrations were varied from 5 to 500 µM with a constant concentration of 2 mM ATP. Alternately, ATP concentrations were varied from 5 to 100 µM with a constant 2 mM concentration of AMP.

An alternate assay for the confirmation of the ADP reaction product was the FPLC analysis of the reaction mixture. Assay mixtures contained 25 mM TES/K+ buffer pH 7.0, 5 mM MgCl2, 10 mM DTT, 1mM ATP, 1 mM AMP and AdkB (1 g) in a final volume of 1 ml. Alternately, 2 mM ADP was added in place of ATP/AMP in order to observe the formation of ATP and AMP. Reaction mixtures were incubated at 70 C for 30 minutes and 500 µl of the reaction mixture was injected onto a MonoQ HR column (1 x 8 cm, Amersham Bioscience) with a linear gradient from 0-1 M NaCl in 25 mM TES pH 7.5 over 55 mL at 1 mL/min. Nucleotides were detected by absorbance at 280 nm. Under these conditions AMP, ADP, and ATP eluted at 25, 30, and 33 minutes, respectively.

Temperature Stability ofAdkB.The temperature stability of AdkB was determined by incubating 1 g enzyme in assay buffer (100 L) at 70, 80, 90 or 100 C for 10 minutes in sealed tubes. Following heating, the enzyme mixture was cooled on ice, centrifuged to return all contents to the bottom of the tube, and substrates were added. The final composition and volume of the reaction mixture was as described for the standard assay above.

References:

1.Graham, D. E., Xu, H., and White, R. H. (2002) Identification of coenzyme M biosynthetic phosphosulfolactate synthase: a new family of sulfonate biosynthesizing enzymes, J. Biol. Chem.277, 13421-13429.

2.Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem.72, 248-254.

3.Adams, H. (1963) Adenosine-5'-diphosphate and Adenosine-5'-monophosphate, In Methods of Enzymatic Analysis (Bergmeyer, H. U., Ed.), pp 573-577, Academic Press, New York.

4.Barzu, O., Abrudan, I., Proinov, I., Kiss, L., Ty, N. G., Jebeleanu, G., Goia, I., Kezdi, M., and Mantsch, H. H. (1976) Nucleotide specificity of pyruvate kinase and phosphoenolpyruvate carboxykinase, Biochim. Biophys. Acta.452, 406-412.

5.Plowman, K. M., and Krall, A. R. (1965) A kinetic study of nucleotide interactions with pyruvate kinase, Biochemistry4, 2809-2814.

Supplemental Figure 1

N-terminal Sequence Alignment for Bacterial and Archaeal UMP kinases, (UK, green shading), the new Archaeal Type II AMP kinases(AdkB, blue shading), and the Bacterial and Archaeal Type I AMP kinases (AdKA, red shading). The respective KxSG, KxGG and GxxGxG(S/G), GxxGxGK motifs are indicated. Sequences used in the alignment are as follows: E.coli.-Escherichia coli O157;BA1797-Bacillus anthracis str. Ames; MJ1259-Methanocaldococcus jannaschii; SSO0976-Sulfolobus solfataricus; MJ0458-Methanocaldococcus jannaschii;.MmarC70644-Methanococcus maripaludis C7; Mbur1317-Methanococcoides burtonii (strain DSM 6242)MJ0479-Methanocaldococcus jannaschii;PH1753-Pyrococcus horikoshii OT3;E.coli.AdkA (EcE220899)-Escherichia coli E22.

E.coli.Uk -----MATNAKPVYKRILLKLSGEALQGTEGFGIDASILDRMAQEIKELVELGIQVGVVI 55

BA1797.Uk -----MRP-----YKRVLIKLSGGALADQTGNSFNSKRLEHIANEILSIVDLGIEVSIVI 50

MJ1259.Uk MILITLLKDGVGEKMRIVFDLGGSVVMPKEGA--KAEKIMEYANIFKKIKDEGHEVAIVV 58

SSO0976.UK ------MNIILKISGKFFDED-----NVDNLIVLRQSIKELADNGFRVGIVT 41

MJ0458.AdkB ------MHIVKIGGSLTYDA------KPLLKALKN--YAKENNKKIVIIP 36

MmarC7_0644.AdkB ------MVIVYVVKIGGSLTYNA------EKLLESLK------QSCEKIVIIP 35

Mbur1317.AdkB ------MKVVLKLGGSLIDRS------SELIKAISDHFAATEGNMQVIIVP 39

MJ0479.AdkA ------MKNKVVVIVGVPGVGS------TTVTNKAIEELKKEGIEYKIVN 38

PH1753.AdkA ------MP-FVVIITGIPGVGK------STITKLALQRTR---AKFKLIN 34

E.coli.AdkA ------MRIILLGAPGAGKG----TQAQFIMEKYGIPQISTGDMLRAAVK 40