Molecular, Biochemical, and Functional Characterization of a Nudix Hydrolase Protein That Stimulates the Activity of a Nicotinoprotein Alcohol Dehydrogenase
2002; Elsevier BV; Volume: 277; Issue: 38 Linguagem: Inglês
10.1074/jbc.m205617200
ISSN1083-351X
AutoresHarm Kloosterman, Jan W. Vrijbloed, Lubbert Dijkhuizen,
Tópico(s)Polyamine Metabolism and Applications
ResumoThe cytoplasmic coenzyme NAD+-dependent alcohol (methanol) dehydrogenase (MDH) employed by Bacillus methanolicusduring growth on C1-C4 primary alcohols is a decameric protein with 1 Zn2+-ion and 1–2 Mg2+-ions plus a tightly bound NAD(H) cofactor per subunit (a nicotinoprotein). Mg2+-ions are essential for binding of NAD(H) cofactor in MDH protein expressed in Escherichia coli. The low coenzyme NAD+-dependent activity of MDH with C1–C4 primary alcohols is strongly stimulated by a second B. methanolicus protein (ACT), provided that MDH contains NAD(H) cofactor and Mg2+-ions are present in the assay mixture. Characterization of the act gene revealed the presence of the highly conserved amino acid sequence motif typical of Nudix hydrolase proteins in the deduced ACT amino acid sequence. Theact gene was successfully expressed in E. coli allowing purification and characterization of active ACT protein. MDH activation by ACT involved hydrolytic removal of the nicotinamide mononucleotide NMN(H) moiety of the NAD(H) cofactor of MDH, changing its Ping-Pong type of reaction mechanism into a ternary complex reaction mechanism. Increased cellular NADH/NAD+ratios may reduce the ACT-mediated activation of MDH, thus preventing accumulation of toxic aldehydes. This represents a novel mechanism for alcohol dehydrogenase activity regulation. The cytoplasmic coenzyme NAD+-dependent alcohol (methanol) dehydrogenase (MDH) employed by Bacillus methanolicusduring growth on C1-C4 primary alcohols is a decameric protein with 1 Zn2+-ion and 1–2 Mg2+-ions plus a tightly bound NAD(H) cofactor per subunit (a nicotinoprotein). Mg2+-ions are essential for binding of NAD(H) cofactor in MDH protein expressed in Escherichia coli. The low coenzyme NAD+-dependent activity of MDH with C1–C4 primary alcohols is strongly stimulated by a second B. methanolicus protein (ACT), provided that MDH contains NAD(H) cofactor and Mg2+-ions are present in the assay mixture. Characterization of the act gene revealed the presence of the highly conserved amino acid sequence motif typical of Nudix hydrolase proteins in the deduced ACT amino acid sequence. Theact gene was successfully expressed in E. coli allowing purification and characterization of active ACT protein. MDH activation by ACT involved hydrolytic removal of the nicotinamide mononucleotide NMN(H) moiety of the NAD(H) cofactor of MDH, changing its Ping-Pong type of reaction mechanism into a ternary complex reaction mechanism. Increased cellular NADH/NAD+ratios may reduce the ACT-mediated activation of MDH, thus preventing accumulation of toxic aldehydes. This represents a novel mechanism for alcohol dehydrogenase activity regulation. Methanol is formed in large quantities in mineralization processes in nature, mostly from degradation of methylesters and -ethers that occur in plants (pectin and lignin). Methylotrophic microorganisms growing on methanol as carbon and energy sources can be isolated readily from soil. They possess a special set of enzymes for generation of energy from methanol oxidation and for synthesis of compounds with carbon–carbon bonds from methanol (1Anthony C. The Biochemistry of Methylotrophs. Academic Press, London1982Google Scholar, 2Dijkhuizen L. Rehm H.J. Reed G. Puhler A. Stadler P. Bio/Technology. VCH Publishers, Weinheim, Germany1993: 265-284Google Scholar). Three different type of enzymes catalyze the initial oxidation of methanol to formaldehyde in methylotrophs. Yeasts employ an alcohol oxidase with FAD as cofactor (a flavoprotein); oxygen is used as electron acceptor, resulting in hydrogen peroxide formation. This enzyme is located in peroxisomes, an organel that also contains catalase activity (3Harder W. Veenhuis M. Rose A.H. Harrison J.S. The Yeasts. Academic Press, London1989: 289-316Google Scholar). Gram-negative bacteria employ a methanol dehydrogenase (MDH) 1The abbreviations used are: MDH, methanol dehydrogenase; bMDH, MDH purified from B. methanolicus cells; cMDH, MDH purified from E. colicells; Nudix, nucleotide diphosphate group linked to some other moiety (x); ORF, open reading frame; ADPR, ADP-ribose. with pyrroloquinoline quinone as cofactor (a quinoprotein), located in the periplasmic space (4Anthony C. Adv. Microb. Physiol. 1986; 27: 113-210Crossref PubMed Scopus (204) Google Scholar, 5Duine J.A. Frank J. Jongejan J.A. FEMS Microbiol. Rev. 1986; 32: 165-178Crossref Google Scholar). Gram-positive bacteria (bacilli and actinomycetes) employ cytoplasmic MDH enzymes with NAD(P)+as cofactor (nicotinoproteins), constituting novel NAD(P)+-dependent alcohol dehydrogenases with unusual properties (6Reid M.F. Fewson C.A. Crit. Rev. Microbiol. 1994; 20: 13-56Crossref PubMed Scopus (355) Google Scholar, 7Bystrykh L. Arfman N. Dijkhuizen L. Murrell J.C. Kelly D.P. Microbial Growth on C1 Compounds. Intercept Ltd., Andover, MA1993: 245-251Google Scholar, 8Hektor H.J. Kloosterman H. Dijkhuizen L. J. Mol. Cat. B. 2000; 8: 103-109Crossref Scopus (20) Google Scholar). Pure cultures of obligately aerobic, thermotolerant Bacillus methanolicus strains grow rapidly in methanol mineral medium (doubling times 40–80 min) at temperatures of 35–60 °C and are tolerant to very high methanol concentrations (9Arfman N. Dijkhuizen L. Kirchhof G. Ludwig W. Schleifer K.H. Bulygina E.S. Chumakov K.M. Govorukhina N.I. Trotsenko Y.A. White D. Sharp R.J. Int. J. Syst. Bacteriol. 1992; 42: 439-445Crossref PubMed Scopus (66) Google Scholar, 10Dijkhuizen L. Arfman N. Attwood M.M. Brooke A.G. Harder W. Watling E.M. FEMS Microbiol. Lett. 1988; 52: 209-214Crossref Scopus (37) Google Scholar). All strains studied employ a coenzyme NAD+-dependent MDH for growth on methanol and other primary alcohols (C1–C4) (11de Vries G.E. Arfman N. Terpstra P. Dijkhuizen L. J. Bacteriol. 1992; 174: 5346-5353Crossref PubMed Google Scholar, 12Arfman N. Watling E.M. Clement W. van Oosterwijk R.J. de Vries G.E. Harder W. Attwood M.M. Dijkhuizen L. Arch. Microbiol. 1989; 152: 280-288Crossref PubMed Scopus (70) Google Scholar). MDH from B. methanolicus strain C1 belongs to family III of alcohol dehydrogenases (11de Vries G.E. Arfman N. Terpstra P. Dijkhuizen L. J. Bacteriol. 1992; 174: 5346-5353Crossref PubMed Google Scholar, 13Vonck J. Arfman N. de Vries G.E. Van Beeumen J. van Bruggen E.F. Dijkhuizen L. J. Biol. Chem. 1991; 266: 3949-3954Abstract Full Text PDF PubMed Google Scholar). It is a decameric enzyme with subunits of 43,000 Da (10Dijkhuizen L. Arfman N. Attwood M.M. Brooke A.G. Harder W. Watling E.M. FEMS Microbiol. Lett. 1988; 52: 209-214Crossref Scopus (37) Google Scholar, 11de Vries G.E. Arfman N. Terpstra P. Dijkhuizen L. J. Bacteriol. 1992; 174: 5346-5353Crossref PubMed Google Scholar, 13Vonck J. Arfman N. de Vries G.E. Van Beeumen J. van Bruggen E.F. Dijkhuizen L. J. Biol. Chem. 1991; 266: 3949-3954Abstract Full Text PDF PubMed Google Scholar, 14Arfman N. de Vries K.J. Moezelaar H.R. Attwood M.M. Robinson G.K. van Geel M. Dijkhuizen L. Arch. Microbiol. 1992; 157: 272-278Crossref PubMed Scopus (19) Google Scholar, 15Dijkhuizen L. Arfman N. FEMS Microbiol. Rev. 1990; 87: 215-220Crossref Scopus (5) Google Scholar, 16Arfman N. Van Beeumen J. de Vries G.E. Harder W. Dijkhuizen L. J. Biol. Chem. 1991; 266: 3955-3960Abstract Full Text PDF PubMed Google Scholar, 17Arfman N. Hektor H.J. Bystrykh L.V. Govorukhina N.I. Dijkhuizen L. Frank J. Eur. J. Biochem. 1997; 244: 426-433Crossref PubMed Scopus (45) Google Scholar). Each MDH subunit contains 1 Zn2+-ion and 1–2 Mg2+-ions (13Vonck J. Arfman N. de Vries G.E. Van Beeumen J. van Bruggen E.F. Dijkhuizen L. J. Biol. Chem. 1991; 266: 3949-3954Abstract Full Text PDF PubMed Google Scholar). Zinc is commonly found in the active site of alcohol dehydrogenases, but the presence of magnesium had not been reported before. Each subunit also contains a tightly (but noncovalently) bound NAD(H) molecule, which is oxidized and reduced by methanol and formaldehyde, respectively, and thus functions as a cofactor (17Arfman N. Hektor H.J. Bystrykh L.V. Govorukhina N.I. Dijkhuizen L. Frank J. Eur. J. Biochem. 1997; 244: 426-433Crossref PubMed Scopus (45) Google Scholar). NAD+ plays two important roles in the MDH-catalyzed reaction: the MDH-bound NAD+cofactor serves as the primary electron acceptor in the alcohol dehydrogenase reaction, and exogenous NAD+ coenzyme is responsible for reoxidation of the MDH-bound NADH cofactor. MDH obeys a Ping-Pong type of reaction mechanism, consistent with such a temporary parking of reducing equivalents at the MDH-bound NAD(H) cofactor (17Arfman N. Hektor H.J. Bystrykh L.V. Govorukhina N.I. Dijkhuizen L. Frank J. Eur. J. Biochem. 1997; 244: 426-433Crossref PubMed Scopus (45) Google Scholar). This is very different from typical NAD+coenzyme-dependent alcohol dehydrogenases, which follow a ternary complex type of reaction mechanism (6Reid M.F. Fewson C.A. Crit. Rev. Microbiol. 1994; 20: 13-56Crossref PubMed Scopus (355) Google Scholar). Studies with purified proteins showed that MDH activity with C1–C4 primary alcohols and its affinity for exogenous NAD+ and alcohol substrates are strongly increased in the presence of a B. methanolicus strain C1-soluble M r 50,000 activator (ACT) protein. Activation, which takes place within 1 s upon the addition of saturating amounts of ACT, requires the presence of Mg2+-ions. Spectral studies showed that Mg2+-ions are essential for the formation of an MDH·ACT·NAD+·Mg2+ complex. At physiological methanol concentrations (0.1–1.0 mm), the methanol turnover rate of MDH in vitro was increased up to 40-fold by the ACT protein (16Arfman N. Van Beeumen J. de Vries G.E. Harder W. Dijkhuizen L. J. Biol. Chem. 1991; 266: 3955-3960Abstract Full Text PDF PubMed Google Scholar). Synthesis of the MDH and ACT proteins in B. methanolicus is regulated coordinately (14Arfman N. de Vries K.J. Moezelaar H.R. Attwood M.M. Robinson G.K. van Geel M. Dijkhuizen L. Arch. Microbiol. 1992; 157: 272-278Crossref PubMed Scopus (19) Google Scholar). Here we report a molecular, biochemical, and functional characterization of the ACT protein of B. methanolicus strain C1. ACT is a member of the Nudix hydrolase family, that display hydrolytic activity with substrates containing anucleotide diphosphate group linked to some other moiety (x) (18Bessman M.J. Frick D.N. O'Handley S.F. J. Biol. Chem. 1996; 271: 25059-25062Abstract Full Text Full Text PDF PubMed Scopus (589) Google Scholar). ACT protein stimulates coenzyme NAD+-dependent MDH activity by hydrolytic removal of the NMN(H) part of the NAD(H) cofactor in MDH protein. B. methanolicus strain C1 cells were grown as described (11de Vries G.E. Arfman N. Terpstra P. Dijkhuizen L. J. Bacteriol. 1992; 174: 5346-5353Crossref PubMed Google Scholar). Escherichia coli strains MC1061 and JM109, serving as hosts for genetic modifications and heterologous gene expression, respectively, were grown on LB medium (19Sambrook J. Frisch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar), and when appropriate, ampicillin (100 mg.l−1) was added. The primers used were: A1, 5′-GGCGAATTCAA(A/G)TT(A/G)TT(T/C)GA(A/G)GA(A/G)AA(A/G)AC-3′, and A2, 5′-GGCTGATCATC(C/ T/A)AC(T/C)TG(T/C)AA(T/C)TT(C/T/A) AC(C/T/A)AC-3′. These were based on the N-terminal amino acid sequence of the ACT protein (16Arfman N. Van Beeumen J. de Vries G.E. Harder W. Dijkhuizen L. J. Biol. Chem. 1991; 266: 3955-3960Abstract Full Text PDF PubMed Google Scholar). Their degree of degeneration was limited based on codon bias of B. methanolicus (11de Vries G.E. Arfman N. Terpstra P. Dijkhuizen L. J. Bacteriol. 1992; 174: 5346-5353Crossref PubMed Google Scholar). Primer A3 consisted of an EcoRI site followed by the first 19 nucleotides of the act gene: 5′-GCGGAATTCATGGGAA- AATTATTTGAGG-3′. The antisense primer A4 contained a BamHI site and the last 18 nucleotides ofact: 5′-CGCGGATCCTCATTTATGTTTGAGAGC-3′. The KS and SK primers used are available commercially (Stratagene, Westburg, Leusden, The Netherlands). Primer positions are shown in Fig.1. DNA amplification reactions were performed with Vent-DNA polymerase (New England Biolabs). Reaction mixtures (100 ml) contained: enzyme buffer (New England Biolabs), dNTP (50 mm/nucleotide), primers (0.5 mm/primer), target DNA (1–10 ng). Target DNA was incubated for 5 min at 94 °C before adding polymerase. Amplification conditions were: 25 reaction cycles at temperatures for denaturation, primer annealing and primer extension of 94, 45, and 72 °C, respectively. In the last reaction cycle, the primer extension time was set at 5 min. Plasmid pHK1 was constructed by cloning the PCR product of primers A1/A2, with B. methanolicus chromosomal DNA as template, in pBlueScriptIIKS (Stratagene). Plasmid pHK83 contains a 4.4-kb insert of B. methanolicus chromosomal DNA in thePstI site of pBlueScriptIIKS. Plasmid pHK105 was constructed by cloning the act gene in the EcoRI and BamHI sites of pProk1 (CLONTECH Laboratories, Palo Alto, AC) with act expression controlled by the pProk1 tac promoter. B. methanolicus strain C1 total chromosomal DNA was isolated as described (11de Vries G.E. Arfman N. Terpstra P. Dijkhuizen L. J. Bacteriol. 1992; 174: 5346-5353Crossref PubMed Google Scholar). Methods for DNA handling, modification, and cloning were performed as described previously (19Sambrook J. Frisch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). Chromosomal DNA of B. methanolicus was digested withPstI, and fragments ranging from 3.7 to 5.1 kb were isolated, ligated in pBlueScriptIIKS, and transformed to E. coli MC1061. This partial gene library sequence was screened using primers A1/A2. 40 × 250 pooled E. coli MC1061 colonies were transferred from agar plates to liquid LB medium and incubated for 4 h at 37 °C. Samples from these 40 cultures were lysed by boiling for 5 min and subjected to DNA amplification. Cultures giving a positive amplification signal were plated, and 400 colonies were screened in pools of 20 colonies. Finally, individual colonies were screened for a positive amplification signal. Nucleotide sequencing was done using dye-primers in the cycle sequencing method (20Murray V. Nucleic Acids Res. 1989; 17: 8889Crossref PubMed Scopus (438) Google Scholar) with the Thermosequenase kit RPN 2538 fromAmersham Biosciences. The samples were run on the ALF-Express sequencing robot. Analysis of the nucleotide sequence (deposited in the GenBankTM data base under accession number AY128667) was done using CloneManager, Version 4.01. Protein sequence comparisons were performed using the facilities of the BLAST server (21Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (70758) Google Scholar) at NCBI (National Library of Medicine, Washington, D. C.). Cells were disrupted by two passages through a French pressure cell operating at 1.4 × 105kilonewtons.m−2. Enzyme assays were performed at 50 °C with prewarmed buffer solutions (unless stated otherwise). The NAD+-dependent MDH activity and MDH-stimulating activity of ACT were measured as described earlier (12Arfman N. Watling E.M. Clement W. van Oosterwijk R.J. de Vries G.E. Harder W. Attwood M.M. Dijkhuizen L. Arch. Microbiol. 1989; 152: 280-288Crossref PubMed Scopus (70) Google Scholar, 16Arfman N. Van Beeumen J. de Vries G.E. Harder W. Dijkhuizen L. J. Biol. Chem. 1991; 266: 3955-3960Abstract Full Text PDF PubMed Google Scholar). Assays were performed at 50 °C, and NAD(H) oxidation or reduction was followed at 340 nm in a Hitachi model 100-60 spectrophotometer. The MDH assay, buffered by 100 mm glycine-KOH buffer (pH 9.5), contained 5 mm MgSO4, 5 mm2-mercaptoethanol, 1 mm NAD+, and enzyme. The reaction was started with 500 mm methanol after a 5-min preincubation. The stimulatory effect of activator protein was analyzed by the subsequent addition of purified activator protein. One unit of MDH-stimulating activity is defined as the amount of ACT that stimulates a fixed quantity of purified MDH (1.0 μg (2.5 pmol) MDH/ml reaction mixture) to 50% of fully ACT-activated MDH (V max) minus the ACT-independent activity of MDH (V o) (16Arfman N. Van Beeumen J. de Vries G.E. Harder W. Dijkhuizen L. J. Biol. Chem. 1991; 266: 3955-3960Abstract Full Text PDF PubMed Google Scholar). Nudix hydrolase activity of ACT protein was determined in 300 μl of 100 mm glycine-KOH buffer (pH 9.5), containing 10 mm MgSO4 and 250 nmol (di-)nucleotide at 50 °C. Dinucleotide hydrolyzing activity was assayed using an excess amount of calf intestine alkaline phosphatase (EC 3.1.3.1, Roche Molecular Biochemicals), to hydrolyze the nucleoside monophosphates formed to nucleosides and orthophosphate. Alternatively, in assays with mononucleotides as substrate, yeast inorganic pyrophosphatase (EC3.6.1.1, Roche Molecular Biochemicals) was added to hydrolyze pyrophosphate to orthophosphate. After a 5-min preincubation, reactions were started with 2.5 μg (59 pmol) of purified activator protein. After 10 min the reactions were terminated with 350 μl of 1n H2SO4. Orthophosphate was determined by a slight modification of the method of Ames and Dubin (22Ames B.N. Dubin D.T. J. Biol. Chem. 1960; 235: 769-775Abstract Full Text PDF PubMed Google Scholar); 350 ml of a 2× concentrated ascorbate-molybdate mixture was added. After a 20-min incubation at 45 °C, the absorbance of the solution was read in a Hitachi model 100–60 spectrophotometer at 820 nm. 8-oxo-dGTP, which is not commercially available, was prepared as described (23Mo J.Y. Maki H. Sekiguchi M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11021-11025Crossref PubMed Scopus (299) Google Scholar). Enzyme kinetics was studied using standard assay conditions and varying substrate conditions. Data were fitted with Sigma Plot for Windows, Version 5.0 (Jandell Scientific Software) according to the Michaelis-Menten equation. Data obtained in the MDH-activator protein titration experiments was fitted according to the Hill equation. ACT protein was purified from an 8-liter batch culture ofE. coli JM109 (pHK105) (Table I). Cells were grown to an A 595 of 0.5, 1 mmisopropyl β-d-thiogalactopyranoside was added, and heterologous gene expression was allowed for a further 2 h, yielding a final A 595 of 1.35. Cells were harvested by centrifugation (25 min, 6,500 × g).Table IPurification of ACT protein from E. coli JM109 (pHK105)Purification stepTotal proteinSpecific activityYieldPurification factormgunits.mg−1%Crude extract5344251001.0Heat treatment1401232762.9Q-Sepharose671746514.1Superdex-200531638383.9Mono-Q362301375.4One unit of MDH-stimulating activity is defined as the amount of ACT that stimulates a fixed quantity of purified MDH (1.0 μg; 2.5 pmol) in the reaction mixture (1 ml) to 50% of V max− V o under reaction conditions described (16Arfman N. Van Beeumen J. de Vries G.E. Harder W. Dijkhuizen L. J. Biol. Chem. 1991; 266: 3955-3960Abstract Full Text PDF PubMed Google Scholar). Open table in a new tab One unit of MDH-stimulating activity is defined as the amount of ACT that stimulates a fixed quantity of purified MDH (1.0 μg; 2.5 pmol) in the reaction mixture (1 ml) to 50% of V max− V o under reaction conditions described (16Arfman N. Van Beeumen J. de Vries G.E. Harder W. Dijkhuizen L. J. Biol. Chem. 1991; 266: 3955-3960Abstract Full Text PDF PubMed Google Scholar). Crude extract (9.8 ml containing 534 mg of protein) was prepared as described above. Crude extract was incubated for 30 min at 60 °C. Denatured proteins were precipitated by centrifugation for 10 min at 25,000 × g. The pellet was washed thoroughly with buffer A (20 mm Tris-HCl pH 7.5, 5 mm MgSO4, 5 mm 2-mercaptoethanol) to recover any ACT protein present in the pellet. The preparation obtained (6.9 ml) contained 140 mg of protein. The sample was loaded onto a Q-Sepharose column equilibrated with buffer A. Proteins were eluted by applying a 40-ml linear 0–1 m KCl gradient at a flow rate of 1 ml.min−1. ACT peak fractions were pooled yielding a preparation (12 ml) containing 67 mg of protein. 3-ml samples were applied onto a Superdex-200 column equilibrated with buffer A at a flow rate of 2 ml.min−1. ACT peak fractions of four separate runs were combined yielding a preparation (12 ml) with 53 mg of protein. 2-ml samples were applied onto a Mono-Q column equilibrated with buffer A. Proteins were eluted by applying a 20-ml linear 0–1 m KCl gradient at a flow rate of 0.5 ml.min−1. Pooled fractions with ACT activity of six separate runs were combined and frozen at −80 °C. The final preparation (13 ml) contained 36 mg of protein. MDH was purified from B. methanolicus andE. coli as described (12Arfman N. Watling E.M. Clement W. van Oosterwijk R.J. de Vries G.E. Harder W. Attwood M.M. Dijkhuizen L. Arch. Microbiol. 1989; 152: 280-288Crossref PubMed Scopus (70) Google Scholar) with some modifications. Overnight cultures were harvested by centrifugation, and cells were disrupted by two passages through a French Pressure cell at 140 megapascals. Crude extracts were prepared by centrifugation for 30 min at 40,000 × g. Proteins were partially precipitated by 30% saturation with ammonium sulfate and incubation for 10 min. Following centrifugation (10 min at 25,000 × g) the supernatant was applied on a Phenyl-Superose (hydrophobic interaction) column equilibrated with 20% (w/v) (NH4)2SO4 in buffer A (50 mm Tris/HCl, 5 mm MgSO4, 5 mm β-mercaptoethanol, pH 7.5). Proteins were eluted with a gradient of 20–0% (w/v) (NH4)2SO4. Active fractions were pooled, desalted on PD-10 columns, and applied on a Mono-Q (anion exchange) column. Proteins were eluted with a 1–0 m KCl gradient in buffer A. To separate ACT, MDH containing the bound NAD(H) cofactor, and coenzyme NAD(H), reaction mixtures were loaded on a Phenyl-Superose hydrophobic interaction column equilibrated with 20% (w/v) (NH4)2SO4 in buffer A (50 mm Tris/HCl, 5 mm MgSO4, 5 mm β-mercaptoethanol, pH 7.5). Free nucleotides (NAD+ and NADH) appeared in the flow-through, whereas proteins were eluted with a gradient of 20–0% (w/v) (NH4)2SO4. ACT eluted at 11% (NH4)2SO4 and MDH at 5% (NH4)2SO4. A standard MDH reaction was performed in a total volume of 10 ml. The reaction mixture contained 100 mm glycine-KOH buffer (pH 9.5), 5 mm MgSO4, 5 mm2-mercaptoethanol, 500 mm methanol, 1 mmNAD+, 1.0 nmol (400 μg) of pure MDH (10 nmol subunits), and in the activated MDH reaction, 2.5 nmol (105 μg) of pure ACT. The reaction was started by the addition of MDH protein and terminated in a few seconds by rapid freezing in liquid nitrogen. MDH protein was separated from free nucleotides with a Phenyl-Superose hydrophobic interaction column and desalted on a Pharmacia PD-10 column equilibrated with 10 mm Tris-HCl, pH 8.0, containing 6m urea (buffer B). The desalted protein fraction was boiled for 2 min and applied onto a Mono-Q anion exchange column equilibrated with buffer B and eluted in a gradient of 0–1 m KCl in buffer B. A solution of AMP and NADH (10 nmol each) prepared in buffer B and boiled for 2 min served as references on the Mono-Q column. The metal composition of purified MDH was determined by atomic absorption spectrophotometry using a PerkinElmer 1100B atomic absorption spectrophotometer. Prior to analysis, the enzyme was dialyzed extensively against 10 mm Tris-HCl buffer (pH 7.5). The elements magnesium and zinc were analyzed in duplicate. Use of the A1/A2 primers based on the N-terminal amino acid sequence of ACT yielded a PCR fragment of the expected size (89 bp), which was cloned into pBlueScriptIIKS (pHK1) and sequenced. The deduced amino acid sequence was in full compliance with the previously determined N terminus of the ACT protein (16Arfman N. Van Beeumen J. de Vries G.E. Harder W. Dijkhuizen L. J. Biol. Chem. 1991; 266: 3955-3960Abstract Full Text PDF PubMed Google Scholar), indicating that the correct DNA fragment had been amplified. Southern hybridization experiments with different digests of chromosomal DNA ofB. methanolicus strain C1, using the cloned PCR fragment as probe, revealed in all cases only one clear hybridizing signal (results not shown). A 4.4-kb PstI DNA fragment that hybridized well with the probe was selected to be cloned. For that purpose a partial gene library sequence of B. methanolicus chromosomal DNA was constructed in pBlueScriptIIKS (insert frequency, 60%). Using the primers A1 and A2, a total of 10,000 E. coli MC1061 transformants were screened, yielding 16 PCR-positive transformants. Plasmid DNA analysis of the positive transformants showed that all contained a 4.4-kb PstI insert (pHK83). In total, 1480 bp of the 4.4-kb PstI fragment in plasmid pHK83 was sequenced in both directions, revealing the presence of two open reading frames. Theact ORF (from ATG381 to TGA935, 558 bp in size) encodes a putative protein of 185 amino acids with Mr 21,048. A potential ribosome binding site (AGGA) was identified immediately upstream ofact. The act ORF is immediately followed by ORF2 (at least 545 bp). Screening of the available data bases revealed a strong similarity between ACT and the YQKG gene product of Bacillus subtilis (P54570, 62% identity and 83% similarity with the full-length protein), encoding an ADP-ribose pyrophosphatase (24Dunn C.A. O'Handley S.F. Frick D.N. Bessman M.J. J. Biol. Chem. 1999; 274: 32318-32324Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). The highly conserved sequence motif characteristic for the E. coli MutT-related proteins or Nudix hydrolase family was detected in the deduced ACT amino acid sequence (Fig.2). Extracts of E. coli MC1061 containing pHK83 failed to stimulate MDH activity. Clear act gene expression in E. coli was observed following its introduction in the expression vector pProk1, yielding pHK105. After induction with isopropyl β-d-thiogalactopyranoside, extracts of E. coliJM109 (pHK105) displayed high ACT protein activities (425 units.mg−1 protein of MDH-stimulating activity). ACT activity in its native host, B. methanolicus, was estimated as 8.6 units.mg−1 (16Arfman N. Van Beeumen J. de Vries G.E. Harder W. Dijkhuizen L. J. Biol. Chem. 1991; 266: 3955-3960Abstract Full Text PDF PubMed Google Scholar). SDS-PAGE revealed the presence of a Mr 21,000 protein in strain JM109 (pHK105) that was absent in strain JM109 carrying pProk1. ACT protein was estimated to constitute about 40% of the total soluble protein fraction. Following induction with isopropyl β-d-thiogalactopyranoside, cell extracts were prepared from 8-liter cultures of E. coli JM109 (pHK105).B. methanolicus is a thermotolerant bacterium, able to grow at temperatures up to 60 °C (9Arfman N. Dijkhuizen L. Kirchhof G. Ludwig W. Schleifer K.H. Bulygina E.S. Chumakov K.M. Govorukhina N.I. Trotsenko Y.A. White D. Sharp R.J. Int. J. Syst. Bacteriol. 1992; 42: 439-445Crossref PubMed Scopus (66) Google Scholar, 15Dijkhuizen L. Arfman N. FEMS Microbiol. Rev. 1990; 87: 215-220Crossref Scopus (5) Google Scholar), and we observed that ACT protein in E. coli extracts is stable at this temperature for at least 2 h. E. coli proteins started to denature after ∼10 min at 60 °C. After 30 min of incubation, denatured proteins were collected by centrifugation and discarded. A combination of anion exchange chromatography and gel filtration steps allowed purification of active ACT protein. The Mrof native ACT protein was estimated as 45,000 by gel filtration chromatography on a Superdex-200 column. ACT thus is a homodimer of 21-kDa subunits. SDS-PAGE showed that this procedure yielded pure ACT protein, with a final purification factor of 5.4 and a total yield of 37% (Table I). ACT appears to be a thermostable enzyme; at 100 °C the half-life of its MDH stimulating activity is 1.5 min. The ACT protein concentration required for maximal stimulation of in vitroactivity of MDH purified from B. methanolicus (bMDH) was determined (Fig. 3). The addition of 1.0 μg.ml−1 (24 pmol.ml−1) ACT protein stimulated initial MDH activity rates (1.0 μg.ml−1 (2.5 pmol.ml−1) MDH protein) by ∼10-fold. The presence of Mg2+-ions was a prerequisite for this stimulating effect (Fig. 3). In view of the presence of the highly conserved Nudix hydrolase sequence motif in ACT, its (di-)nucleotide hydrolyzing activity was evaluated. Experiments were performed in a glycine-KOH buffer (pH 9.5) at 50 °C, the pH and temperature values optimal for MDH activity and for the stimulatory effect of ACT on MDH (16Arfman N. Van Beeumen J. de Vries G.E. Harder W. Dijkhuizen L. J. Biol. Chem. 1991; 266: 3955-3960Abstract Full Text PDF PubMed Google Scholar). No activity was detected with the canonical (deoxy-)nucleoside triphosphates, 8-oxo-dGTP, or with diadenosine tri- or tetraphosphates, previously shown to be substrates for members of the Nudix hydrolase family (18Bessman M.J. Frick D.N. O'Handley S.F. J. Biol. Chem. 1996; 271: 25059-25062Abstract Full Text Full Text PDF PubMed Scopus (589) Google Scholar). However, incubations of ACT with ADP-ribose (ADPR) revealed that this is an outstanding substrate for the enzyme (V max, 348 μmol.min−1.mg−1;K m, 63 μm). ACT also showed a clear NAD+ hydrolyzing activity, although the substrate affinity and V max for NAD+ are much lower, resulting in a catalytic efficiency (k cat/K m) of 3–4 orders of magnitude lower than for ADPR (0.48 × 103 versus 2.05 × 106m−1.s−1) (Fig.4, TableII). The presence of Mg2+-ions is a prerequisite for ACT catalyzed ADPR and NAD+ hydrolysis. ACT did not show any NADP(H) hydrolyzing activity. Only a very low NADH hydrolyzing activity was detected, at least a factor 100 lower than the NAD+ hydrolysis rate. AMP plus ribose 5′-phosphate and AMP plus NMN+, were identified as the ACT hydrolysis products of ADPR and NAD+, respect
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