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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Biochemistry. Author manuscript; available in PMC 2010 June 30.
Published in final edited form as:
PMCID: PMC2757067
NIHMSID: NIHMS121388

Biochemical Characterization of the GTP:Adenosylcobinamide-phosphate Guanylyltransferase (CobY) Enzyme of the Hyperthermophilic Archaeon Methanocaldococcus jannaschii

Abstract

The archaeal cobY gene is the non-orthologous replacement of the bacterial NTP:AdoCbi kinase (EC 2.7.7.62) / GTP:AdoCbi-P guanylyltransferase (EC 3.1.3.73) and is required for de novo synthesis of AdoCbl (coenzyme B12). Here we show that ORF MJ1117 of the hyperthermophilic, methanogenic archaeon Methanocaldococcus jannaschii encodes a CobY protein that transfers the GMP moiety of GTP to AdoCbi-P to form AdoCbi-GDP. Results from isothermal titration calorimetry (ITC) experiments show that MjCobY binds GTP (Kd = 5 µM), but it does not bind the GTP analogs GMP-PNP (guanosine 5'-(β,γ)-imidotriphosphate) or GMP-PCP (guanylyl 5'-(β,γ)-methylenediphosphonate), nor GDP. Results from ITC experiments indicate that MjCobY binds one GTP per dimer. Results from in vivo studies support the conclusion that the 5’-deoxyadenosyl upper ligand of AdoCbi-P is required for MjCobY function. Consistent with these findings, MjCobY displayed high affinity for 5’-adenosyl moiety of AdoCbi-P (Kd = 0.76 µM), but did not bind non-adenosylated Cbi-P; kinetic parameters for theMj CobY reaction were determined. We propose that ORF MJ1117 of Methanocaldococcus jannaschii be annotated as cobY to reflect its involvement in AdoCbl biosynthesis. Results from circular dichroism studies indicate that, in isolation, MjCobY denatures at 80ºC with a concomitant loss of activity.

Keywords: Coenzyme B12 biosynthesis, archaeal metabolism, corrinoid enzymology, nucleotide loop assembly, coenzymes of methanogenic archaea

Cobamides are ancient cofactors that are widely distributed in nature (1). With the exception of plants, cells of all domains of life have enzymes that require a cobamide as their coenzyme, yet only some bacteria and archaea synthesize cobamides de novo (24).

Although not all cobamide producers synthesize the corrin ring de novo, some can salvage pre-formed, incomplete precursors [e.g., cobinamide (Cbi), cobyric acid (Cby)] from their environments using a high- affinity ATP-binding cassette transporter (510). Precursors such as Cbi and Cby are then converted to cobamides by two distinct branches of the pathway. One of these branches, known as the corrinoid adenosylation pathway, attaches the upper axial ligand to the corrin ring via a labile C-Co bond (11). The second branch assembles the nucleotide loop that tethers the lower ligand base to the corrin ring; this branch is known as the nucleotide loop assembly pathway (Fig. 1). The latter can be further broken down into two sub-branches, one of which activates adenosyl-Cbi (AdoCbi) to AdoCbi-guanosine diphosphate (AdoCbi-GDP) (12), and a second one that activates the lower ligand base to its nucleotide (2) (Fig. 1).

Figure 1
Nucleotide loop assembly pathway

In bacteria, the activation of AdoCbi to AdoCbi-GDP is catalyzed by the bifunctional CobU enzyme (EC 2.7.1.156, EC 2.7.7.62) via an AdoCbi-P intermediate (13). Archaea, however, use a different strategy for the synthesis of AdoCbi-GDP (2). These organisms do not synthesize CobU; instead, they use CobY, a GTP:AdoCbi-P guanylyltransferase enzyme that lacks the NTP:AdoCbi-P kinase activity of CobU (Fig. 1) (14). The cobY gene was identified in extreme halophilic and thermophilic, methanogenic archaea as a non-orthologous replacement for cobU (14, 15).

In this work we identified the locus encoding the CobY enzyme in the hyperthermophilic methanogenic archaeon Methanocaldococcus jannaschii. Recombinant protein was expressed in E. coli cells, the order of substrate binding was determined, and the interactions between substrate and enzyme were quantified.

EXPERIMENTAL PROCEDURES

Microbiological Techniques

Bacteria, culture media, and growth conditions

Bacterial strains and plasmids used in these studies are listed in Table 1. Salmonella enterica strains were cultured in nutrient broth (NB; Difco); E. coli strains were cultured in lysogeny broth (LB) (16, 17). Plasmids were maintained by the addition of ampicillin, (100 µg/mL), chloramphenicol (20 µg/mL), kanamycin (50 µg/mL), or tetracycline (20 µg/mL) to NB or LB. No-carbon essential (NCE) medium (18) supplemented with glycerol (30 mM) and MgSO4 (1 mM) was used to grow cells under nutrient-defined conditions. Solid media contained 15 g of Bacto Agar (Difco) per liter. Corrinoids [cyanocobyric acid (CNCby), dicyanocobinamide [(CN)2Cbi], or cyanocobalamin [CNCbl; a.k.a. vitamin B12] were added to media at 15 nM. CNCby was a gift from Paul Renz (Institut für Biologische Chemie und Ernahrungswissenschaft, Universität-Hohenheim, Stuttgart, Germany); (CN)2Cbi and CNCbl were purchased from Sigma.

Table 1
Strains and plasmids used in these studies.

Genetics and Recombinant DNA Techniques

Complementation of NTP:AdoCbi-P nucleotidyltransferase activity in Salmonella enterica ΔcobU ΔycfN strain

Plasmid pCOBY14 (cobY+) was electroporated (19)into strain JE8268 (ΔcobU ΔycfN) (20) to assess complementation of AdoCbi-P nucleotidyltransferase activity; the resulting strain was JE8335 (Table 1). Plasmid pT7-7 was used as negative control in complementation experiments and was moved into strain JE8268 by electroporation, yielding strain JE8269. Isolated colonies of strains JE8268, JE8269, JE8335, and TR6583 were inoculated into NB and incubated at 37°C overnight (~16 h). Five-µl aliquots of the overnight cultures were diluted into 195 µl of NCE-glycerol minimal medium as described above and incubated at 37°C with shaking in a 96-well microtiter plate using an ELx808 Ultra Microplate Reader (Bio-Tek Instruments).

Strain construction

Construction of a ΔcobS strain of Salmonella enterica. A chromosomal in-frame deletion of the cobS gene was constructed in strain TR6583 as described (21), using primers OL14 (5’-ATG AGT AAG CTG TTT TGG GCC ATG CTC GCT TTT ATT AGC CGC TTG CCC GTG GTG TAG GCT GGA GCT GCT TC-3’) and OL24 (5’-TCA TAA CAG AGC CAG CAG AAA GAT CAA TTC ACC AAG TTC GAT CGC CGC GCC GAC CGT ATC GCC GGT TTG ACC GCC CAT ATG AAT ATC CTC CTT AG −3’); the resulting strain was JE8248. Deletion of the cobs gene was verified by DNA sequencing using BigDye® protocols (ABI PRISM); reaction mixtures were resolved by the UW-Madison Biotechnology Center.

Construction of a strain deficient in the attachment of the upper ligand. Corrinoid adenosylation was blocked by inactivation of the cobA gene encoding the housekeeping ATP:corrinoid adenosyltransferase (11). The cobA363::MudJ mutation was introduced into the chromosome of strain JE8268 (ΔcobU ΔycfN) by bacteriophage P22-mediated transduction (22).

Biochemical Techniques

Protein overexpression and purification

CobY protein. Twenty mL of LB supplemented with ampicillin and chloramphenicol were inoculated with E. coli strain BL21-CodonPlus®(DE3)RIL (Stratagene) harboring plasmid pCOBY14 (M.j. cobY+) and incubated at 37°C overnight. One liter of LB supplemented with ampicillin was inoculated with the overnight culture and incubated at 37°C with shaking at 200 rpm until the culture density reached OD600 of 0.5 to 0.6. Isopropyl-β-D-1-thiogalactopyranoside (IPTG) was added to 0.5 mM to induce expression of cobY+. To obtain maximum yield of the recombinant protein, incubation was continued for approximately 16 h at 37°C. Cells were harvested by centrifugation at 4°C at 15,000×g for 15 min using a JA-25.50 rotor in an Avanti™ J-25I Beckman/Coulter refrigerated centrifuge and resuspended in 15 mL of tris(hydroxymethyl)aminomethane hydrochloride buffer (Tris-HCl; 100 mM, pH 8.0 @ 4°C) containing protease inhibitor phenylmethylsulphonyl fluoride (1 mM). Cells were placed on ice and lysed by sonication for 2 min (5-s pulse followed by 10 s of cooling) in a model 550 sonic dismembrator (Fisher). The extract was cleared by centrifugation at 4°C for 30 min at 43,367×g. The cell-free extract was heated in a water bath at 75°C for 15 min, and precipitated E. coli proteins were cleared by centrifugation at 4°C for 60 min at 43,367×g. Finely ground ammonium sulfate (UltraPure, ICN Biomedicals) was added to 10% (w/v.) Precipitates were cleared by centrifugation at 4°C for 60 min at 43,367×g.

CobY protein was purified in two steps from clarified extract via fast protein liquid chromatography (FPLC) using an ÄKTAprime system (GE Healthcare).

Step 1: Hydrophobic interaction chromatography. Cell-free extract was applied to a 5-mL HiTrap Phenyl (high-sub) FF column (GE Healthcare) equilibrated with 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid buffer (HEPES; 50mM, pH 7.5 @ 4°C) containing (NH4)2SO4 (10% w/v.) Protein was eluted at a flow rate of 2.5 mL min−1 with a linear gradient to 100% HEPES buffer, (50 mM; pH 7.5 @ 4°C.) The CobY protein was found in the flow-through fractions, which were concentrated using a Centricon® 70 centrifugal filter device (Millipore) and dialyzed against HEPES buffer, (50 mM; pH 7.5 @ 4°C) to remove (NH4)2SO4.

Step 2: Ion exchange chromatography. The concentrated protein solution was applied to a 1-mL MonoQ 5/50 column (GE Healthcare) equilibrated with HEPES buffer, (50mM; pH 7.5 @ 4°C.) Protein was eluted at a flow rate of 1 mL min−1 with a linear gradient to 100% HEPES buffer, (50mM; pH 7.5 @ 4°C) containing NaCl (1M.) CobY did not interact with the resin, and flow-through fractions containing CobY were concentrated and dialyzed against HEPES buffer (50mM; pH 7.5 @ 4°C). Glycerol was added to a final concentration of 10% (v/v). The CobY protein was flash-frozen by drop-wise addition into liquid N2; frozen beads were stored at −80°C until use.

Salmonella enterica CobS protein. Cell-free membrane fractions enriched for SeCobS were prepared as reported (23).

Assessment of protein concentration and purity

Protein concentration was determined by the Bradford method (24). Purity was assessed by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (25), followed by staining with Coomassie Brilliant Blue R-250 (26). Protein purity was established by band densitometry using a computer-controlled Fotodyne imaging system with Foto/Analyst v.5.00 software (Fotodyne Incorporated) for image acquisition and TotalLab® v.2005 software for analysis (Nonlinear Dynamics).

Size exclusion chromatography

Size exclusion chromatography was performed as described (27) except that the molecular weight standards used were 159.5, 34.6, 11.3, and 1.9 kDa. A standard curve was created in order to calculate the molecular mass of each peak from its elution time; log molecular mass (in kDa) was linear between 25 min and 42 min elution (r2 = 1.000).

Synthesis of corrinoid substrates

AdoCbi and AdoCbi-P were prepared and purified as reported (20).

in vitro synthesis of Adocobalamin-5’-phosphate (AdoCbl-P): CobY-CobS coupled reaction

Reactions were performed under dim, red light to avoid photolysis of the C-Co bond of the adenosylated corrinoid substrate. Reaction mixtures contained Tris-HCl (50 mM, pH 7.9), α-ribazole-5′-phosphate (α-RP; 30 µM) (28), MgCl2 (5 mM), GTP (2 mM), 5 µg of CobS-enriched membrane extract or membrane material from a control strain in which cobS was not overexpressed (23), CobY protein (460 pmol), and AdoCbi-P (0.2 mM) in a final volume of 20 µl. Reaction mixtures were incubated at 37°C for 1 h. Reactions were stopped and corrinoids converted to their cyanated forms by the addition of 3 µl of KCN (100 mM), heating at 80°C for 10 min., and irradiation with a 60-W incandescent light at a distance of 6 cm for 15 min. Complete cobamides were detected using a bioassay as described (20) and identified by reverse-phase high-performance liquid chromatography (RP-HPLC) and mass spectrometry (see below).

In vitro synthesis of AdoCbi-GDP by CobY

Reaction mixtures contained Tris-HCl (50 mM, pH 7.9), GTP (2 mM,) NaCl (50 mM), MgCl2 (10 mM), dithiothreitol (DTT; 10 mM), MjCobY protein (2.3 pmol), and AdoCbi-P (200 µM) in a final volume of 40 µl. Reaction mixtures were incubated under dim, red light for 15 min. Reactions were stopped and corrinoids converted to their cyanated forms as described above.

HPLC analysis of the MjCobY reaction product

Fifty-five µl of water were added to the MjCobY reaction mixtures, which were then filtered using Spin-X centrifugal filters (Corning Costar). Corrinoid reactants and products were separated by RP-HPLC on a System Gold HPLC system (Beckman Coulter) equipped with an Alltima HP C18 AQ 5 µm 150 mm×4.6 mm column fitted with an Alltima HP C18 AQ All-Guard cartridge (Alltech). The column was equilibrated with a buffer system of 77% A-23% B (see below). A 14.4-min linear gradient was applied at 1 ml min−1 until the composition of the buffer system was 67% A-33% B. The solvents used were: buffer A [100 mM potassium phosphate buffer (pH 6.5) containing KCN (10 mM)]; buffer B [100 mM potassium phosphate buffer (pH 8.0) containing 10 mM KCN]:CH3CN (1:1)]. Corrinoids eluted from the column were detected at 367 nm with a Waters photodiode array detector. A standard curve for (CN)2Cbi was constructed for quantification purposes; detection of (CN)2Cbi was linear between 1 pmol and 3 nmol (r2 = 0.9984).

Mass spectrometry

The product of the MjCobY-SeCobS coupled reaction was dried under vacuum in a SpeedVac® concentrator (Thermo Savant,) suspended in ddH2O, and loaded onto a Sep-pak C18 cartridge (Waters) pre-equilibrated with ddH2O. Corrinoids were eluted from the resin with 2.5 mL of 100% (v/v) methanol, and the sample was dried under vacuum. The dried sample was analyzed at the Mass Spectrometry Facility at the University of Wisconsin-Madison Biotechnology Center. Mass spectra were obtained using a Bruker Daltronics (Billerica, MA) BILFLEX III matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometer.

Kinetic analyses

Kinetic parameters were determined using the assay conditions described above, using Microsoft® Excel software.

Isothermal titration calorimetry

MjCobY protein was dialyzed against Tris-HCl buffer, pH 8.0 at 4°C (50mM) containing NaCl (50 mM,) MgCl2 (10 mM,) and tris(2-carboxyethyl)phosphine hydrochloride (4 mM); GTP and AdoCbi-P were dissolved in spent dialysis buffer. Experiments were conducted at 27°C and consisted of 27 10-µl injections of a 1 mM stock of ligand into a 1.45-mL sample cell containing 18µM CobY dimers. Injections were made over a period of 20 s with a 3-min interval between injections. The sample cell was stirred at 307 rpm. Data were acquired on a VP-ITC Microcalorimeter (Microcal™, Northhampton, MA) and analyzed with the ORIGIN® v.7.0383 software provided with the microcalorimeter.

Circular dichroism spectroscopy

CobY protein was dialyzed against sodium phosphate buffer, pH 7.2 (50mM,) containing NaCl (50mM) and submitted for circular dichroism (CD) spectroscopy to the Biophysics Instrumentation Facility, Department of Biochemistry, University of Wisconsin-Madison. Protein concentration for CD spectroscopy was measured on a SoloVPE variable pathlength extension spectrophotometer (C Technologies, Inc., Bridgewater, NJ) using an extinction coefficient of 14,900 M−1 cm −1 and determined to be 0.2 mg/mL. CD spectra were recorded on a Model 202SF Circular Dichroism Spectrometer (Aviv Biomedical, Lakewood, NJ.) The cell pathlength was 0.1 cm for CD measurements. CD spectra were recorded from 300 to 200 nm at 1 nm steps with an averaging time of 5 s for baselines and 3 s for protein-containing samples. Baselines were recorded at 20, 37 and 80°C and used as baselines for the respective temperatures.

Results and Discussion

Identification of the gene encoding CobY in Methanocaldococcus jannaschii

The MJ1117 open reading frame (ORF) of M. jannaschii encodes a protein that has been shown to catalyze the guanylylation of an intermediate in the synthesis of coenzyme F420 in methanogenic archaea (29). Because the chemistry of the F420 biosynthetic reaction is similar to that of the guanylylation of AdoCbi-P (30), we hypothesized that the MJ1117 protein catalyzed the synthesis of AdoCbi-GDP from AdoCbi-P and GTP in M. jannaschii.

Bioinformatics analyses support the assignment of the MJ1117 gene product as the NTP:AdoCbi-P nucleotidyltransferase enzyme in M. jannaschii. Using the protein Basic Local Alignment Search Tool (BLASTP) program from the National Center for Biotechnology Information (31), we searched the M. jannaschii proteome for homologues of CobY proteins from Methanothermobacter thermautotrophicus (Mth; formerly Methanobacterium thermoautotrophicum) strain ΔH (14) and Halobacterium sp. strain NRC-1 (15). ORF MJ1117 encoded a putative protein that shared 34% identity and 58% similarity to Mth CobY (Expect = 6e-26,) and 31% identity and 55% similarity to the CobY protein of Halobacterium sp. NRC-1 (Expect = 6e-08). Although the level of identity was low, we suspected that ORF MJ1117 encoded the M. jannaschii CobY protein; hence, hereafter we refer to ORF MJ1117 as M.j. cobY.

In vivo evidence that MjCobY has NTP:AdoCbi-P nucleotidyltransferase activity

The CobY proteins studied to date lack AdoCbi kinase activity. To determine whether MjCobY had AdoCbi kinase and/or AdoCbi-P nucleotidyltransferase activity, we introduced plasmid pCOBY14 (M.j. cobY+) into S. enterica strain JE8268 (ΔcobU ΔycfN) (20). This strain lacks both AdoCbi kinase and AdoCbi-P nucleotidyltransferase activity; thus, it can neither synthesize AdoCbl de novo nor convert AdoCbi or AdoCby to AdoCbl. The ability of strain JE8268 to convert AdoCby to AdoCbl was restored by the addition of the M.j. cobY+ gene in trans (Fig. 2A), indicating that MjCobY functioned as an NTP:AdoCbi-P nucleotidyltransferase in vivo. Growth was not restored in medium supplemented with (CN)2Cbi (data not shown,) indicating that MjCobY lacked NTP:AdoCbi kinase activity.

Figure 2
Cbl-dependent growth of S. enterica strains

CobY activity requires an adenosylated corrinoid substrate

To determine whether MjCobY could use non-adenosylated corrinoid substrate in vivo, we blocked the conversion of Cby to AdoCby by CobA, the housekeeping ATP:corrinoid adenosyltransferase (11). A null allele of the cobA gene (cobA363::MudJ) was introduced into the chromosome of S. enterica strain JE8268 (ΔcobU ΔycfN) by bacteriophage P22-mediated transduction (22). Kanamycin-resistant transductants were freed of phage (22), and plasmid pCOBY14 (M.j. cobY+) was introduced by electroporation (19). Cbl-dependent growth of the resulting strain was assessed to determine whether MjCobY would use non-adenosylated Cby. Only AdoCby supported growth of the transductants, suggesting that MjCobY required adenosylated corrinoid substrate in vivo (Fig. 2B).

In vitro evidence that CobY has GTP:AdoCbi-P guanylyltransferase activity

The ΔcobU ΔycfN strain failed to salvage exogenous AdoCbi-GDP (the product of the MjCobY reaction) (data not shown), leaving unanswered the question of whether MjCobY had GTP:AdoCbi-P guanylyltransferase activity. To address this question we isolated recombinant MjCobY protein (Fig. 3A) and took three in vitro approaches to determine whether MjCobY made AdoCbi-GDP. First, we coupled the MjCobY reaction to the SeCobS enzyme (cobalamin-5'-phosphate synthase; EC 2.7.8.26) and used a bioassay to look for formation of AdoCbl-P (the product of SeCobS) (23). We used strain JE8248 (ΔcobS) (23) as the indicator strain to detect the presence of AdoCbl-P in the reaction mixture. Strain JE8248 is a Cbl auxotroph whose growth requires Cbl or Cbl-P. Conditions for the bioassay have been described (20). The product of the coupled MjCobY-SeCobS reaction supported growth of strain JE8248, while the reaction mixture that did not contain CobY did not (data not shown.) Secondly, we converted the corrinoid product of the MjCobY reaction and detected it by RP-HPLC (Fig. 4A). Thirdly, the purified, cyanated corrinoid product of the MjCobY-SeCobS reaction was analyzed by mass spectrometry. The MALDI-TOF mass spectrum of the reaction product contained a molecular ion signal with an m/z of 1409.4512 amu, consistent with a [Cbl-P]+ ion. Additional signals for [Cbi-GDP]+ and [(CN)Cbi-GDP]+ ions (m/z = 1436.4041 and 1452.3751, respectively) were also observed (Fig. 4B).

Figure 3
Isolation of MjCobY
Figure 4
The product of the MjCobY reaction is AdoCbi-GDP

Interactions of CobY with its substrates

We used isothermal titration calorimetry (ITC) to obtain information about enzyme:substrate binding stoichiometry, to identify the preferred nucleotide and corrinoid substrates for MjCobY, to determine the order of substrate binding, and to quantify the thermodynamic parameters of the interactions between MjCobY and its substrates.

CobY functions as a dimmer

Binding to GTP was observed when the protein concentration used in the calculation of thermodynamic parameters was adjusted to reflect dimeric, rather than monomeric, protein (n = 0.99 ± .01) (Fig. 5.) Size exclusion chromatography revealed that the purified protein was actually a mix of monomers and dimers (Fig. 3B). Oligomers were separated and each species found to be stable when passed separately over the size exclusion column, i.e., dimers did not separate to monomers or vice versa. MjCobY dimers were able to bind both GTP and AdoCbi-P (see below); dimers were used for all subsequent in vitro experiments.

Figure 5
CobY binds GTP

GTP is the nucleotide substrate of MjCobY

Previous to this work, the preferred nucleotide substrate of CobY was not known. Results from ITC experiments revealed that MjCobY bound GTP (Fig. 5, Table 2) but failed to bind other NTPs, the GTP analogs GMP-PNP (5’-guanylylimidotriphosphate) or GMP-PCP (guanylyl 5'-(β,γ)-methylenediphosphonate,) or GDP (data not shown,) strongly suggesting that GTP is the nucleotide substrate for the enzyme in vivo. Favorable enthalpy changes [ΔH = (−1.70 ± 0.02)×104 kcal/mol] drove the binding of GTP to MjCobY. The binding constant (Kb) for GTP was (2.00 ± 0.11)×105 M−1, which corresponded to a dissociation constant (Kd) of 5.0 ± .02 µM. Table 2 also shows the thermodynamic parameters for association of GTP-bound MjCobY and AdoCbi-P.

Table 2
Thermodynamic parameters of the interactions of CobY with its substrates.

The adenosyl upper ligand of the corrinoid substrate is required for binding

Although we showed that MjCobY required an adenosylated corrinoid substrate in vivo and in vitro, it was unclear whether the adenosyl moiety was required for binding to the enzyme, for catalysis, or for both. To examine the role of the upper ligand, we used ITC to determine the enthalpic change of binding of MjCobY to corrinoids with different upper ligands, including AdoCbi-P, (CN)2Cbi-P, and hydroxycobinamide-phosphate [(HO)Cbi-P]. MjCobY did not bind either (CN)2Cbi-P or (HO)Cbi-P (data not shown,) indicating that the upper ligand was important for binding. At present it is unclear whether the adenosyl group is involved in catalysis.

CobY must bind GTP before binding AdoCbi-P

ITC experiments showed that MjCobY would not bind AdoCbi-P in the absence of GTP (Fig. 6,) indicating that the enzyme binds GTP first, before binding AdoCbi-P.

Figure 6
CobY binds GTP before binding AdoCbi-P

Kinetic parameters of the MjCobY reaction

We used RP-HPLC to quantify the in vitro formation of AdoCbi-GDP and to determine pseudo-first order kinetic parameters of the MjCobY reaction at 37°C. Under conditions of saturating GTP concentration (2 mM) and varying AdoCbi-P levels, Kmapp = 18.4 ± 1.1 µM, kcat = 4.7 ± 0.5 s−1, and kcat/Kmapp = 2.6 × 105 M−1 s−1. Under conditions of saturating corrinoid concentration (100 µM) and varying GTP levels, the apparent Km (Kmapp) = 2.4 ± 0.3 µM, kcat = 1.8 ± 0.2 s−1, and the catalytic efficiency (kcat/Kmapp) = 7.5 × 105 M−1 s−1. The lower kcat when GTP was at sub-saturating levels suggestsed that the enzyme must be fully saturated with GTP for maximum turnover. To calculate the maximal reaction rate (Vmax), the reaction mixture contained MjCobY (2.3 pmol), AdoCbi-P (100 µM), and GTP (2 mM). Under the assay conditions used, Vmax = 1.36 ± 0.1 µmol min−1 mg−1 .

When experiments were repeated at 80°C, we found that neither the Kmapp nor the kcat differed appreciably from those obtained when the experiments were conducted at 37°C (data not shown.) This result was surprising, given that: i) M. jannaschii grows optimally at 85°C; and ii) its rapid doubling time of 26 min at that temperature (32) would require substantial amounts of Cba for energy generation, thus demanding high efficiency of Cba biosynthetic enzymes. We used circular dichroism (CD) spectroscopy to examine whether temperature influenced the secondary structure of the MjCobY protein. A 40-min incubation at 80°C resulted in a time-dependent decrease in intensity, indicating that the protein denatured at 80°C (Fig. 7A). In contrast, a 30-min incubation at 37°C did not affect the conformation of the protein. Notably, a shift from 80 ºC to 20ºC did not reverse the conformational change back to the one observed for the protein at 37 ºC (Fig.7B). This suggests that the lack of increase in enzyme activity at 80°C was due to partial denaturation of the MjCobY protein at the elevated temperature. It is possible that, in vivo, the MjCobY protein is stabilized by interactions with other proteins or cell structures, examples of which have been reported in the literature (3336).

Figure 7
Temperature-induced changes in MjCobY conformation

Grochowski et al. recently showed that MjCobY catalyzed the condensation of GTP and 2-phospho-L-lactate (LP) during coenzyme F420 biosynthesis, in a reaction analogous to the guanylylation of AdoCbi-P (37). However, MjCobY’s high Kmapp for LP (6 mM), as compared to that for AdoCbi-P (18.4 µM), is inconsistent with its role in coenzyme F420 biosynthesis. In addition, the authors identified locus MJ0887 as encoding the kinetically competent lactoyl phosphate guanylyltransferase (CofC) enzyme. Nevertheless, structural data would help us understand how MjCobY binds GTP and is able to use both AdoCbi-P and LP as substrates in vitro. This work is currently in progress.

We have shown here that the M. jannaschii MJ1117 gene product is a GTP:AdoCbi-P guanylyltransferase in vivo and in vitro. Based on the data presented, we propose a change in gene nomenclature, from MJ1117 to cobY, to reflect the role of the MjCobY protein in AdoCbl biosynthesis.

Acknowledgments

This work was supported by funds from the NIH to J.C.E.-S. (GM40313). We thank Robert H. White (Virginia Polytechnic Institute and State University) for providing the M.j. cobY+ plasmid for over-expression. We also thank Paul Renz for his gift of cyanocobyric acid.

Abbreviations

MjCobY
Methanocaldococcus jannaschii CobY protein
Cbl
cobalamin
Cbl-P
Cbl-phosphate
AdoCbl
adenosyl-Cbl
AdoCbl-P
adenosyl-Cbl-phosphate
Cbi
cobinamide
(CN)2Cbi
dicyano-Cbi
HOCbi
hydroxy-Cbi
HOCbi-P
HOCbi-phosphate
CNCby
cyanocobyric acid
α-RP
α - ribazole-5’-phosphate
Tris-HCl
tris(hydroxymethyl)aminomethane hydrochloride
HEPES
4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid
Cba
cobamide
FPLC
fast protein liquid chromatography
RP-HPLC
reverse-phase high performance liquid chromatography
ITC
isothermal titration calorimetry
CD
circular dichroism
NTP
nucleoside triphosphate
TCEP
tris(2-carboxyethyl)phosphine

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