Biochem. J. (2007) 408, 327–333 (Printed in Great Britain)
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doi:10.1042/BJ20071017
A CBS domain-containing pyrophosphatase of Moorella thermoacetica is regulated by adenine nucleotides 1 ¨ Joonas JAMSEN* , Heidi TUOMINEN*1 , Anu SALMINEN*, Georgiy A. BELOGUROV*2 , Natalia N. MAGRETOVA†, Alexander A. BAYKOV†3 and Reijo LAHTI*3
*Department of Biochemistry and Food Chemistry, University of Turku, FIN-20014 Turku, Finland, and †A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119899, Russia
CBS (cystathionine β-synthase) domains are found in proteins from all kingdoms of life, and point mutations in these domains are responsible for a variety of hereditary diseases in humans; however, the functions of CBS domains are not well understood. In the present study, we cloned, expressed in Escherichia coli, and characterized a family II PPase (inorganic pyrophosphatase) from Moorella thermoacetica (mtCBS-PPase) that has a pair of tandem 60-amino-acid CBS domains within its N-terminal domain. Because mtCBS-PPase is a dimer and requires transition metal ions (Co2+ or Mn2+ ) for activity, it resembles common family II PPases, which lack CBS domains. The mtCBS-PPase, however, has lower activity than common family II PPases, is potently inhibited by ADP and AMP, and is activated up to 1.6fold by ATP. Inhibition by AMP is competitive, whereas inhibition by ADP and activation by ATP are both of mixed types. The
nucleotides are effective at nanomolar (ADP) or micromolar concentrations (AMP and ATP) and appear to compete for the same site on the enzyme. The nucleotide-binding affinities are thus 100–10000-fold higher than for other CBS-domaincontaining proteins. Interestingly, genes encoding CBS-PPase occur most frequently in bacteria that have a membrane-bound H+ -translocating PPase with a comparable PPi -hydrolysing activity. Our results suggest that soluble nucleotide-regulated PPases act as amplifiers of metabolism in bacteria by enhancing or suppressing ATP production and biosynthetic reactions at high and low [ATP]/([AMP] + [ADP]) ratios respectively.
INTRODUCTION
maximal activity. In addition, bivalent cations, especially Mn2+ , promote dimerization of family II PPases [14]. Interestingly, the N-terminal domain of approx. 25 % of the known family II PPase sequences contains a large (∼ 250-aminoacid) insert, comprising two CBS (cystathionine β-synthase) domains. None of these CBS-PPases has been isolated and characterized. CBS domains, originally found in cystathionine βsynthase [15], are widely distributed among proteins in all three kingdoms of life, but their roles are not well understood [16,17]. In some cases, CBS domains are potential targets for regulation by adenosine derivatives [16–21]. Importantly, point mutations in CBS domains cause several hereditary diseases in humans [17]. To elucidate the role of CBS domains in family II PPases, we have cloned, expressed and purified a CBS-PPase from Moorella thermoacetica (formerly known as Clostridium thermaceticum) (mtCBS-PPase), a low-G + C Gram-positive thermophilic acetogen with an optimum growth temperature of 55–60 ◦C [22]. We found that, unlike all other known PPases, this enzyme is subject to a strong bidirectional regulation by adenine nucleotides.
PPi (inorganic pyrophosphate) is produced in vast amounts by biosynthetic reactions, such as protein, RNA and DNA synthesis, and its concentration affects the equilibria of these reactions [1]. In addition, PPi regulates many other cellular processes, including calcification, cell proliferation and iron transport [2]. Consequently, disruption of PPi metabolism can lead to a variety of pathological conditions [2]. PPi is mainly hydrolysed to Pi (orthophosphate) by PPase (inorganic pyrophosphatase) (EC 3.6.1.1), an enzyme that is essential for life [3–6]. There are two known types of PPase: soluble and integral membrane-bound. Soluble PPases are subdivided further into families I and II, which are not homologous [7,8]. Family I PPases are found in all kingdoms of life and are among the best-characterized phosphoryl transfer enzymes [9,10]. Family II PPases, which were discovered more recently [7,8], are found in Bacilli and Clostridia and in some other bacterial lineages, including several human pathogens, and belong to the DHH (Asp-His-His) family of phosphohydrolases [11]. Family II PPases are homodimers of subunits formed by two well-defined domains, whereas family I PPases have two or six compact one-domain subunits. The N- and C-terminal domains of family II PPases are connected by a flexible linker, and the active site is located at the domain interface [12,13]. Also, unlike family I PPases, family II PPases contain a tightly bound transition metal ion, usually Mn2+ or Co2+ , although they also require Mg2+ for
Key words: adenine nucleotide, bidirectional regulation, cystathionine β-synthetase domain (CBS domain), inorganic pyrophosphatase (PPase), Moorella thermoacetica.
EXPERIMENTAL Cloning and mutagenesis
Genomic DNA extracted from M. thermoacetica strain ATCC 35608 was obtained from DSMZ (Deutsche Sammlung von
Abbreviations used: CBS, cystathionine β-synthase; DTPA, diethylenetriaminepenta-acetic acid; PPase, inorganic pyrophosphatase; mt CBS-PPase, CBS domain-containing PPase from Moorella thermoacetica . 1 These authors contributed equally to this work. 2 Present address: Department of Microbiology and The RNA Group, The Ohio State University, 484 West 12th Avenue, Columbus, OH 43210, U.S.A. 3 Correspondence may be addressed to either of these authors (email
[email protected] or
[email protected]). c The Authors Journal compilation c 2007 Biochemical Society
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Mikroorganismen und Zellkulturen GmbH). The mtCBSPPase open reading frame (GenBank® accession number NC 007644) was amplified by PCR using primers 5 -TTATCATATGGGTAAAGAGATTCTGGTTATCG-3 (forward) and 5 TTATCTCGAGTTATCCCTGCAGCAACCGCCG-3 (reverse). The amplified gene was sequenced in both directions, and one difference compared with the sequence shown in GenBank® was observed (TTG encoding Leu190 was replaced by CTG), but this did not affect the protein sequence. The PCR fragment was inserted via the pET36b vector (Novagen) into pBluescript SK (Stratagene), and site-directed mutagenesis was performed using an overlapping PCR technique (QuikChange® , Stratagene). The mutation was verified by DNA sequencing. Protein expression and purification
To produce wild-type and variant mtCBS-PPases, the PCR products were cloned into the pET36b vector using NdeI and XhoI. The plasmid construct was transformed into Escherichia coli BL21(DE3) RIL cells (Stratagene), and the transformants were grown in Terrific broth [23] containing 30 µg/ml kanamycin and 30 µg/ml chloramphenicol. mtCBSPPase expression was induced for 3 h using 0.4 mM IPTG (isopropyl β-D-thiogalactoside). Cell paste (10 g wet weight) obtained by centrifugation at 6300 g for 10 min was resuspended in 30 ml of ice-cold 25 mM Tris/HCl buffer (pH 7.3), containing 24 mM MgCl2 , 1 mM CoCl2 , 20 µM DTPA (diethylenetriaminepenta-acetic acid), and was homogenized twice using a French press (SLM Instruments) at 900 p.s.i. (1 p.s.i. = 6.9 kPa). The crude extract obtained was loaded on to a 150 ml column containing Fast Flow DEAESepharose (GE Healthcare). The column was washed with the same buffer, and protein was eluted with a 450 ml linear gradient of 0.1–0.3 M NaCl. Fractions containing mtCBS-PPase (150–190 mM NaCl) were pooled, concentrated to 12 ml with Centriprep 10 (Amicon), and purified further by gel filtration on a Superdex 200 26/60 column (GE Healthcare) equilibrated with 50 mM Tris/HCl buffer (pH 7.5), containing 50 mM KCl, 2 mM MgCl2 , 0.1 mM CoCl2 and 20 µM DTPA. The fractions containing mtCBS-PPase were pooled, concentrated to 20– 40 mg/ml and were stored frozen at − 70 ◦C. Because the enzyme was most stable in the presence of 0.1 mM Co2+ and 2 mM Mg2+ , these cations were routinely added to the solutions used to purify, store and dilute the enzyme. When needed, these metal ions were removed from the enzyme stocks by incubation with 5 mM DTPA for 1 day at 4 ◦C. The bulk of the chelator was removed by dialysis against three 1 litre changes of 100 mM Mops/KOH buffer (pH 7.5) containing 50 µM DTPA. The purity of enzyme samples was assessed by electrophoresis on 8–25 % gradient polyacrylamide gels in the presence of 0.55 % SDS using the Phast System (GE Healthcare). Concentrations of mtCBS-PPase solutions were determined on the basis of a subunit molecular mass of 48.1 kDa and a specific absorption coefficient A1% 280 of 4.5, as estimated from the amino acid composition using the ProtParam program [24]. Activity measurements
Except where noted, the activity was measured at 25 ◦C in 25 ml of 100 mM Mops/KOH buffer (pH 7.2) containing 0.1 mM CoCl2 , 5 mM MgCl2 and the nucleotide tested. An aliquot (5–150 µl) of diluted enzyme solution was added to the mixture (0.2–2 µg/ml final enzyme concentration), followed 1 min later by 0.16 mM PPi . The formation of Pi was then monitored for 3–10 min using an automatic Pi analyser [25], and initial reaction rates were estimated from the recorder tracing. c The Authors Journal compilation c 2007 Biochemical Society
AMP, ADP and ATP were from Fluka. All other nucleotides were from Sigma. AMP was the free acid, ADP was the potassium salt, and all other nucleotides were sodium salts. Immediately before the use of ATP, any contaminating ADP was converted into ATP by treating 5 mM stock solutions of ATP for 1 h at ◦ room temperature (23 + − 2 C) with 10 units/ml rabbit muscle creatine kinase (Roche) in 100 mM Mops/KOH buffer (pH 7.2) containing 10 mM phosphocreatine (Fluka) and 5 mM MgCl2 . The AMP preparation was judged to be reasonably pure because a similar treatment with creatine kinase and phosphocreatine had no effect on the PPase activity measured in the presence of AMP. In addition, treating stock ADP solutions with 10 units/ml hexokinase and 10 mM glucose to convert any contaminating ATP into ADP also had no effect on the PPase activity. For competition measurements, in which ADP and ATP were added simultaneously, creatine kinase in stock solutions of ATP was removed by ultrafiltration using 4 ml Vivaspin concentrators (Sartorius AG) with a 10 kDa molecular-mass cut-off PES (polyethersulfone) membrane. Sedimentation
Analytical ultracentrifugation was carried out at 20 ◦C in a Spinco E instrument (Beckman Instruments) with scanning at 280 nm. The samples contained 10 µM enzyme and appropriate ligands. Before each run, the samples were incubated for 2–3 h at 20 ◦C. The sedimentation velocity was measured at 48 000 rev./min, 0 ) was calculated as and the sedimentation coefficient (s20,w described by Chervenka [26]. Cross-linking and electrophoresis
Enzyme was diluted to 8.3 µM with 25 mM Hepes/KOH (pH 7.5) and then incubated with 26 mM glutaraldehyde for 15 or 30 min at room temperature [27]. The reaction was stopped by addition of 1/10 vol. of 1.0 M Tris/HCl (pH 7.3). The samples were separated by electrophoresis on Phast System SDS/8–25 % PAGE gradient gels (GE Healthcare), and the gels were stained with PhastGel Blue R (GE Healthcare). A Perfect Protein Marker kit (Novagen) was used for molecular-mass standards. Kinetic data analysis
Non-linear least squares fitting of the data was performed using SCIENTIST, version 2.01 (Micromath). The dependence of activity on the concentration of the ligand (L) was fitted to eqn (1), where A+L and A−L are activities with and without bound ligand respectively, and K d is the apparent dissociation constant of the enzyme–ligand complex, all measured at a fixed substrate concentration: A = A+L + (A−L − A+L )/(1 + [L]/K d )
(1)
In competition experiments, the dependence of the apparent K d (K d,app ) for ligand L1 on the concentration of the second ligand, L2 , was fitted to eqn (2), where K d1 and K d2 are the dissociation constants for ligands L1 and L2 respectively: K d,app = K d1 (1 + [L 2 ]/K d2 )
(2)
RESULTS Expression, purification and catalytic activity of mt CBS-PPase
SDS/PAGE analysis of crude extracts obtained from recombinant E. coli cells revealed an intense ∼ 50 kDa band that was absent in cells transformed with empty pET36b vector (results not shown).
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Table 1 Effects of nucleotides and nucleosides (100 µM) on Co2+ /Mg2+ activated mt CBS-PPase The activity of mt CBS-PPase measured with no effector added (1.64 s−1 ) was taken as 100 %. Effector
Activity (%)
None Adenosine AMP ADP ATP cAMP CDP GMP GDP UDP UTP Cytidine, guanosine, uridine, CMP, CTP, GTP, cGMP, UMP
100 94 + −2 4.0 + − 0.1
