The Structural Basis for Methylmalonic Aciduria
2004; Elsevier BV; Volume: 279; Issue: 22 Linguagem: Inglês
10.1074/jbc.m401395200
ISSN1083-351X
AutoresV. Saridakis, Alexander F. Yakunin, Xiaohui Xu, Ponni Anandakumar, Micha Pennycooke, Jun Gu, Frederick Cheung, J. Lew, Ruslan Sanishvili, A. Joachimiak, C.H. Arrowsmith, Dinesh Christendat, A.M. Edwards,
Tópico(s)Heme Oxygenase-1 and Carbon Monoxide
ResumoATP:cobalamin adenosyltransferase MMAB was recently identified as the gene responsible for a disorder of cobalamin metabolism in humans (cblB complementation group). The crystal structure of the MMAB sequence homologue from Thermoplasma acidophilum (TA1434; GenBank™ identification number gi|16082403) was determined to a resolution of 1.5 Å. TA1434 was confirmed to be an ATP:cobalamin adenosyltransferase, which depended absolutely on divalent metal ions (Mg2+ > Mn2+ > Co2+) and only used ATP or dATP as adenosyl donors. The apparent Km of TA1434 was 110 μm (kcat = 0.23 s-1) for ATP, 140 μm (kcat = 0.11 s-1) for dATP, and 3 μm (kcat = 0.18 s-1) for cobalamin. TA1434 is a trimer in solution and in the crystal structure, with each subunit composed of a five-helix bundle. The location of disease-related point mutations and other residues conserved among the homologues of TA1434 suggest that the active site lies at the junctions between the subunits. Mutations in TA1434 that correspond to the disease-related mutations resulted in proteins that were inactive for ATP:cobalamin adenosyltransferase activity in vitro, confirming that these mutations define the molecular basis of the human disease. ATP:cobalamin adenosyltransferase MMAB was recently identified as the gene responsible for a disorder of cobalamin metabolism in humans (cblB complementation group). The crystal structure of the MMAB sequence homologue from Thermoplasma acidophilum (TA1434; GenBank™ identification number gi|16082403) was determined to a resolution of 1.5 Å. TA1434 was confirmed to be an ATP:cobalamin adenosyltransferase, which depended absolutely on divalent metal ions (Mg2+ > Mn2+ > Co2+) and only used ATP or dATP as adenosyl donors. The apparent Km of TA1434 was 110 μm (kcat = 0.23 s-1) for ATP, 140 μm (kcat = 0.11 s-1) for dATP, and 3 μm (kcat = 0.18 s-1) for cobalamin. TA1434 is a trimer in solution and in the crystal structure, with each subunit composed of a five-helix bundle. The location of disease-related point mutations and other residues conserved among the homologues of TA1434 suggest that the active site lies at the junctions between the subunits. Mutations in TA1434 that correspond to the disease-related mutations resulted in proteins that were inactive for ATP:cobalamin adenosyltransferase activity in vitro, confirming that these mutations define the molecular basis of the human disease. Cobalamin (vitamin B12) is an important cofactor in both prokaryotes and eukaryotes. A wide variety of cobalamin-dependent metabolic reactions are known, including acetyl-CoA synthesis, generation of deoxyribonucleotides for DNA synthesis, methane production in methanogenic archaea, and fermentation of small molecules (1Roth J.R. Lawrence J.G. Bobbi T.A. Annul. Rev. Microbial. 1996; 50: 137-181Crossref PubMed Scopus (422) Google Scholar). The metabolic requirement for cobalamin varies among different organisms. Some bacteria, such as Escherichia coli, use the vitamin but do not synthesize it (2Dobson C.M. Way T. Lecher D. Nadir H. Nearing M. Lerner-Ellis J.P. Hudson T.J. Rosenblatt D.S. Gravel R.A. Hum. Mol. Genet. 2002; 11: 3361-3369Crossref PubMed Scopus (134) Google Scholar), whereas other bacteria have no need for it (1Roth J.R. Lawrence J.G. Bobbi T.A. Annul. Rev. Microbial. 1996; 50: 137-181Crossref PubMed Scopus (422) Google Scholar). Animals require cobalamin but must rely on dietary sources, and it is believed that plants and fungi neither synthesize nor use cobalamin (1Roth J.R. Lawrence J.G. Bobbi T.A. Annul. Rev. Microbial. 1996; 50: 137-181Crossref PubMed Scopus (422) Google Scholar, 3Martens J.H. Barg H. Warren M.J. Jahn D. Appl. Microbial. Biotechnol. 2002; 58: 275-285Crossref PubMed Scopus (456) Google Scholar). A characteristic that distinguishes cobalamin from all other vitamins is that only certain bacteria and archaea possess the ability for its de novo synthesis (3Martens J.H. Barg H. Warren M.J. Jahn D. Appl. Microbial. Biotechnol. 2002; 58: 275-285Crossref PubMed Scopus (456) Google Scholar, 4Raux E. Schubert H.L. Warren M.J. Cell. Mol. Life Sci. 2000; 57: 1880-1893Crossref PubMed Scopus (162) Google Scholar).Prior to its use in the cell, cobalamin must be metabolized into one of two biologically active forms, adenosylcobalamin (AdoCbl) 1The abbreviations used are: AdoCbl, adenosylcobalamin; HOCbl, hydroxocobalamin; MAD, multiwavelength anomalous diffraction/dispersion; MeCbl, methylcobalamin; r.m.s.d., root mean square deviation.1The abbreviations used are: AdoCbl, adenosylcobalamin; HOCbl, hydroxocobalamin; MAD, multiwavelength anomalous diffraction/dispersion; MeCbl, methylcobalamin; r.m.s.d., root mean square deviation. and methylcobalamin (MeCbl). In humans, these cofactors are known to be used in two enzyme systems. The first enzyme, methionine synthase, uses MeCbl to produce methionine from homocysteine (2Dobson C.M. Way T. Lecher D. Nadir H. Nearing M. Lerner-Ellis J.P. Hudson T.J. Rosenblatt D.S. Gravel R.A. Hum. Mol. Genet. 2002; 11: 3361-3369Crossref PubMed Scopus (134) Google Scholar, 5Harding C.O. Arnold G. Barness L.A. Wolff J.A. Rosenblatt D.S. Am. J. Med. Genet. 1997; 71: 384-390Crossref PubMed Scopus (30) Google Scholar). The second enzyme, methylmalonyl-CoA mutase, uses AdoCbl as a coenzyme to convert methylmalonyl-CoA to succinyl-CoA, which can then enter the trichloroacetic acid cycle (6Leal N.A. Park S.D. Kima P.E. Bobbi T.A. J. Biol. Chem. 2003; 278: 9227-9234Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 7Fenton W.A. Rosenberg L.E. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. 7th Ed. McGraw-Hill Inc., New York1995: 3129-3149Google Scholar). This AdoCbl-dependent reaction is important in the catabolism of methionine, valine, threonine, isoleucine, odd chain fatty acids, and cholesterol (8Drennan C.L. Matthews R.G. Rosenblatt D.S. Ledley F.D. Fenton W.A. Ludwig M.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5550-5555Crossref PubMed Scopus (18) Google Scholar, 9Banerjee R. Chowdhury S. Banerjee J. Chemistry and Biochemistry of B12. John Wiley & Sons, Inc., New York1999: 707-730Google Scholar).Cobalamin itself comprises a central corrin ring that provides four ligands for a hexacoordinated cobalt ion (3Martens J.H. Barg H. Warren M.J. Jahn D. Appl. Microbial. Biotechnol. 2002; 58: 275-285Crossref PubMed Scopus (456) Google Scholar, 4Raux E. Schubert H.L. Warren M.J. Cell. Mol. Life Sci. 2000; 57: 1880-1893Crossref PubMed Scopus (162) Google Scholar). The conversion of cobalamin to AdoCbl and MeCbl involves the reduction of the cobalt ion from a Co(III) oxidation state to Co(I) and then the addition of the functional ligand (2Dobson C.M. Way T. Lecher D. Nadir H. Nearing M. Lerner-Ellis J.P. Hudson T.J. Rosenblatt D.S. Gravel R.A. Hum. Mol. Genet. 2002; 11: 3361-3369Crossref PubMed Scopus (134) Google Scholar, 10Dobson C.M. Way T. Lecher D. Wilson A. Wu X. Dore C. Hudson T. Rosenblatt D.S. Gravel R.A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 15554-15559Crossref PubMed Scopus (118) Google Scholar). In the formation of AdoCbl, the 5′-deoxyadenosyl moiety is transferred from ATP and covalently attached to the Co(I) by an ATP:cobalamin adenosyltransferase (11Fonseca M.V. Escalante-Semerena J.C. J. Biol. Chem. 2001; 276: 32101-32108Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) (Fig. 1). There are three families of adenosyltranserases (EutT, CobA, and PduO) based on amino acid sequences and the pathways with which they are associated.Several inherited diseases are caused by mutations in genes encoding cobalamin metabolic enzymes. These mutations usually cause severe disease early in life, but symptoms can also appear later (12Rosenblatt D.S. Fenton W.A. Scriver C.R. Beaudet A.L. Valle D. Sly W.S. The Metabolic and Molecular Bases of Inherited Disease. 8th Ed. McGraw-Hill, New York2001: 3897-3933Google Scholar). Several different complementation groups are known; cblA, cblB, and cblH have defects related to AdoCbl synthesis, cblE and cblG, have MeCbl defects, and cblC, cblD, and cblF involve synthesis of both cofactors. Depending on the complementation group, patients present with either homocystinuria or methylmalonic aciduria or both. Increased levels of methylmalonic acid are associated with developmental retardation and infant mortality (7Fenton W.A. Rosenberg L.E. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. 7th Ed. McGraw-Hill Inc., New York1995: 3129-3149Google Scholar).The MMAB gene, a PduO-type ATP:cobalamin adenosyltransferase, was recently identified as being responsible for disease associated with the cblB complementation group (2Dobson C.M. Way T. Lecher D. Nadir H. Nearing M. Lerner-Ellis J.P. Hudson T.J. Rosenblatt D.S. Gravel R.A. Hum. Mol. Genet. 2002; 11: 3361-3369Crossref PubMed Scopus (134) Google Scholar, 6Leal N.A. Park S.D. Kima P.E. Bobbi T.A. J. Biol. Chem. 2003; 278: 9227-9234Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar); patients with cblB have methylmalonic aciduria and metabolic ketoacidosis (12Rosenblatt D.S. Fenton W.A. Scriver C.R. Beaudet A.L. Valle D. Sly W.S. The Metabolic and Molecular Bases of Inherited Disease. 8th Ed. McGraw-Hill, New York2001: 3897-3933Google Scholar). cblB patients had mutations in this gene (2Dobson C.M. Way T. Lecher D. Nadir H. Nearing M. Lerner-Ellis J.P. Hudson T.J. Rosenblatt D.S. Gravel R.A. Hum. Mol. Genet. 2002; 11: 3361-3369Crossref PubMed Scopus (134) Google Scholar) and altered protein expression (6Leal N.A. Park S.D. Kima P.E. Bobbi T.A. J. Biol. Chem. 2003; 278: 9227-9234Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). To understand the relationship between the mutations in MMAB and its function, we undertook a biochemical and structural analysis of TA1434, an MMAB sequence homologue from Thermoplasma acidophilum that has 32% sequence identity to MMAB and 35% identity to the PduO adenosyltransferase in Salmonella. Enzyme assays confirmed that TA1434 is indeed an ATP:cobalamin adenosyltransferase, with higher activity than that reported for PduO or CobA. The crystal structure for TA1434 showed that it is a trimer, with each subunit composed of a five-helix bundle. Two disease-related mutations are located in a highly conserved region at the subunit interface, which is the likely active site. In TA1434, mutations that are homologous to disease-causing mutations in the human MMAB resulted in a properly folded yet inactive protein, providing biochemical evidence for the effect of these mutations in cblB patients.EXPERIMENTAL PROCEDURESChemicals—Hydroxocobalamin (HOCbl) was from Sigma. Titanium (III) citrate was prepared as described previously (13Bobbi T.A. Wolfe R.S. J. Bacteriol. 1989; 171: 1423-1427Crossref PubMed Google Scholar). Cloning, Purification, and Crystallographic Studies—Cloning, purification, and crystallization experiments have been described elsewhere (14Christendat D. Saridakis V. Dharamsi A. Bochkarev A. Pai E.F. Arrowsmith C.H. Edwards A.M. J. Biol. Chem. 2000; 275: 24608-24612Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). For the preparation of selenomethionine-enriched protein, ATP: cobalamin adenosyltransferase was expressed in a methionine auxotroph strain B834(DE3) of E. coli and purified under the same conditions as native ATP:cobalamin adenosyltransferase with the addition of 5 mm β-mercaptoethanol in all buffers. The morphology of single crystals of ATP:cobalamin adenosyltransferase is cubic, and the crystals appear after ∼24 h in crystallization set-ups containing ammonium phosphate as the precipitant. The native and selenomethionine-enriched crystals selected for diffraction experiments were grown in 0.4 m ammonium phosphate, 4% methyl-pentanediol, and 5% glycerol at 20 °C. The crystals belonged to the space group P23, with the unit cell parameters a = b = c = 89.1 Å. The Matthew's coefficient, VM, was determined as 3.2 Å3 Da-1, resulting in a solvent content of 61% with a single molecule in each asymmetric unit.X-Ray Diffraction and Structure Determination—A three-wave-length MAD experiment was carried out at 100 K on beamline ID19, Advanced Photon Source, and data from a native crystal of ATP:cobalamin adenosyltransferase were also collected on beamline ID19, Advanced Photon Source. MAD and native data were integrated and scaled with the DENZO/SCALEPACK suite of programs. Data collection statistics are presented in Table I. SOLVE was used to locate the selenium sites and calculate the phases, and RESOLVE was used to modify the density and build a portion of the model. Electron density visualization and model building were done with O. Rigid body and simulated annealing torsion angle refinement were normally followed by individual B-factor refinement and performed using CNS 1.0. Several rounds of refinement were combined with model rebuilding in O after inspection of both 2Fo - Fc and Fo - Fc maps. Refinement statistics are found in Table II.Table IX-ray data collection parameterscAfter SOLVE.X-ray dataNativePeakEdgeRemoteSpace groupP23P23P23P23Resolution (Å)1.551.91.91.9Selenium sites (no.)444Total observations (no.)341,835270,100271,040269,810Unique reflections (no.)62,54035,97735,94635,946Intensity (I/σ⟨I⟩)16 (3.5)26 (3.4)27 (4.0)26 (3.3)Completeness (%)99 (99.8)100 (100)100 (100)100 (100)RsymaRsym=∑|I−⟨I⟩|/∑I, where I is the observed intensity and ⟨I⟩ is the average intensity from multiple observations of symmetry-related reflections.0.094 (0.470)0.080 (0.548)0.076 (0.502)0.081 (0.566)Figure of merit (%)bFigure of merit of phasing=∑P(α)eια|/∑P(α), where P(α) is the phase probability distribution and α is the phase angle.62cAfter SOLVE./72dAfter RESOLVE.a Rsym=∑|I−⟨I⟩|/∑I, where I is the observed intensity and ⟨I⟩ is the average intensity from multiple observations of symmetry-related reflections.b Figure of merit of phasing=∑P(α)eια|/∑P(α), where P(α) is the phase probability distribution and α is the phase angle.c After SOLVE.d After RESOLVE. Open table in a new tab Table IIRefinement parametersNativeRcryst0.197Rfree0.215Protein atoms (no.)1255Water molecules (no.)170R.m.s.d. bond lengths (Å)0.010R.m.s.d. bond angles (°)0.90R.m.s.d. dihedrals (°)17.2Average B-factor (Å2)23.1 Open table in a new tab Gel Filtration—Gel filtration of ATP:cobalamin adenosyltransferase was performed with a Superdex 200 prep 16/60 column (Amersham Biosciences) equilibrated with 10 mm HEPES and 500 mm NaCl using AKTA Explorer (Amersham Biosciences). Protein standards included aldolase, bovine serum albumin, ovalbumin, and cytochrome c. Chromatography was performed at 4 °C at a flow rate of 0.5 ml/min.ATP:Cob(I)alamin Adenosyltransferase Assay—Adenosyltransferase assays were carried out under anaerobic conditions using a modification of a previously published method (15Johnson C.L. Pechonick E. Park S.D. Havemann G.D. Leal N.A. Bobbi T.A. J. Bacteriol. 2001; 183: 1577-1584Crossref PubMed Scopus (103) Google Scholar). The assay mixtures (2 ml) were incubated at 70 °C (or at indicated temperature) under anaerobic conditions in 1-cm glass cuvettes closed by rubber stoppers (Suba Seal) and preflushed with argon for 5 min. The assay components (Master Mix; 200 mm Tris-HCl, pH 8, 0.4 mm ATP, 1.6 mm KH2PO4, and 2.8 mm MgCl2) were sparged with argon for 10 min and transferred anaerobically (using a Hamilton syringe) to the anaerobic spectrophotometric cuvettes. The anaerobic solutions of HOCbl (in methanol; 0.05 mm final concentration) and titanium (III) citrate (1 mm final concentration) were introduced by Hamilton syringes, and after 30 s of argon sparging the cuvettes were incubated at room temperature for 10 min (to allow reduction of HOCbl to cob(I)alamin). After 5 min of equilibration at 70 °C (in a thermo-jacketed cuvette holder), the reactions were initiated by the addition of an anaerobic solution of the enzyme (20-50 μg of protein), and the decrease in absorbance at 388 nm was monitored. The activity was quantified using ϵ = 24.9 mm-1 cm-1.The steady-state kinetic constants for ATP, dATP, and HOCbl were determined under the same conditions as described above except for substrate concentrations. These activity measurements were made for incubation mixtures ranging in substrate concentration from 0.01 to 0.8 mm for ATP (in the presence of 50 μm HOCbl), 0.025 to 1.5 mm for dATP (in the presence of 50 μm HOCbl), and 1 to 50 μm for HOCbl (in the presence of 0.4 mm ATP). All values were in duplicate, and the appropriate S.D. values are given. Data were analyzed with the kinetic analysis program GraphPad Prism® 4 (GraphPad Software Inc., San Diego, CA), and kinetic parameters were obtained by non-linear curve fitting using the saturation kinetic equation v = (Vmax [S])/(Km + [S]).Site-directed Mutagenesis of TA1434 —Site-directed mutagenesis was carried out using QuikChange™ (Stratagene). The mutagenic oligonucleotides used to prepare all of the TA1434 mutants are listed in Table III. DNA encoding wild-type TA1434 cloned into pET15B was used as a template for mutagenesis. Briefly, 25 ng of template DNA was incubated with the appropriate mutagenic primers, dNTPs, and Pfu DNA polymerase using the cycling parameters recommended in the supplier's manual. Following the temperature cycling steps, DpnI was added to each amplification reaction and incubated at 37 °C for 6 h followed by transformation of the mutagenized plasmid into XL2-Blue cells. Plasmid was purified from the resulting colonies using the Qia-prep Spin Mini Prep kit (Qiagen), and all mutations were verified by DNA sequencing.Table IIIATP:Cobalamin adenosyltransferase mutagenic oligonucleotidesMutationOrientationSequenceR119AForward5′-tcg ctc cac atg gca GCG gct gtt tcg agg agg ctt gag-3′Reverse5′-ctc aag cct cct cga aac agc CGC tgc cat gtg gag cga-3′R119WForward5′-tcg ctc cac atg gca TGG gct gtt tcg agg agg ctt gag-3′Reverse5′-ctc aag cct cct cga aac agc CCA tgc cat gtg gag cga-3′R124AForward5′-cgt gct gtt tcg agg GCG ctt gag agg agg ata-3′Reverse5′-tat ctt ctt cac aag CGC cct cga aac agc acg-3′R124FForward5′-cgt gct gtt tcg agg TTT ctt gag agg agg ata-3′Reverse5′-tat cct cct ctc aag AAA cct cga aac agc acg-3′R124KForward5′-cgt gct gtt tcg agg AAA ctt gag agg agg ata-3′Reverse5′-tat cct cct ctc aac TTT cct cga aac agc acg-3′R124WForward5′-cgt gct gtt tcg agg TGG ctt gag agg agg ata-3′Reverse5′-tat ctt ctt ctc aag CCA cct cga aac agc acg-3′E126AForward5′-gtt tcg agg agg ctt GCG agg agg ata gtt gcg-3Reverse5′-cgc aac tat cct cct CGC aag cct cct cga aac-3′E126KForward5′-gtt tcg agg agg ctt AAA agg agg ata gtt gcg-3′Reverse5′-cgc aac tat cct cct TTT aag cct cct cga aac-3′ Open table in a new tab Purification of ATP:Cobalamin Adenosyltransferase Mutants—Expression of the mutants yielded between 10 and 15 mg of soluble, recombinant proteins per liter of cell culture. All of the mutants were purified in the same manner as wild-type ATP:cobalamin adenosyltransferase and appeared as single bands after SDS-PAGE.RESULTS AND DISCUSSIONSequence Analysis—The primary amino acid sequence of the conserved 177-residue protein TA1434 was used to search the NCBI protein data base for sequence homologues. A number of homologues, mostly annotated as hypothetical proteins, were found in archaea, bacteria, and eukaryotes, with sequence identities ranging from 32 to 77%. The ClustalW alignment of TA1434 with eight homologues is shown in Fig. 2. TA1434 has 35% sequence identity to PduO and 32% identity to MMAB, which are ATP:cobalamin adenosyltransferases from Salmonella typhi and humans, respectively, suggesting that TA1434 also has this activity. There are 22 residues that are absolutely conserved among the homologues. The conserved residues Arg-119, Arg-124, and Glu-126 in TA1434 correspond to Arg-186, Arg-191, and Glu-193 in the human MMAB, which are mutated residues in cblB patients (2Dobson C.M. Way T. Lecher D. Nadir H. Nearing M. Lerner-Ellis J.P. Hudson T.J. Rosenblatt D.S. Gravel R.A. Hum. Mol. Genet. 2002; 11: 3361-3369Crossref PubMed Scopus (134) Google Scholar). Because of the relatively weak sequence identity of TA1434 with human MMAB, we first confirmed that TA1434 had ATP:cobalamin adenosyltransferase activity by using an adenosyltransferase assay (shown below) and, thus, we will refer to TA1434 as ATP:cobalamin adenosyltransferase.Fig. 2Partial alignment of TA1434 (tr|Q9HIA7) with sequence homologues. Sequences shown are for the hypothetical protein (HP) TV0097 from Thermoplasma volcanium (tr|Q97CK4), HP SSO0588 from Sulfolobus solfataricus (tr|Q9UWZ1), the YVQK protein from Bacillus subtilis (tr|O34899), HP from Klebsiella pneumoniae (tr|Q48424), PduO from S. typhi (tr|Q8Z5L4), Mus musculus MMAB (sp|Q9D273), Homo sapiens MMAB (sp|Q96EY8), and a putative ATP:cob(I)alamin adenosyltransferase from Porphyromonas gingivalis (tr|Q7MVB4). The boxed amino acids are conserved in an alignment that includes four additional homologues (tr|Q970Z7, tr|Q9KCH6, tr|Q9RTW5, and tr|Q980D7).View Large Image Figure ViewerDownload (PPT)Structure Determination—The structure of ATP:cobalamin adenosyltransferase was determined by the MAD method using selenium as the anomalous scatterer. SOLVE was able to locate four of five selenium sites, and the figure of merit was 62. The resulting electron density was of high quality and allowed the automated model-building segment of RESOLVE to identify and build 124 of 177 amino acids from 30-70, 77-98, and 113-171. Amino acids 71-76 and 99-112 were built manually. The final model consists of 148 residues, from residue 23 to residue 170, and 170 water molecules. The first 22 and the last 7 residues were disordered and could not be built in the electron density map. The model was refined to 1.55 Å resolution with an Rcryst of 0.197 and an Rfree of 0.215. According to PROCHECK, 99.6% of the residues are in the most favored regions, and there are no residues in the disallowed regions of the Ramachandran plot (16Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar).Structure Overview—ATP:cobalamin adenosyltransferase consists of a five-helix bundle (helices α1, α2, α3, α4, and α5) (Fig. 3A). Helix α1 extends from residue 25 to residue 45, helix α2 continues from residue 48 to residue 71, helix α3 runs from residues 80 to 97, helix α4 extends from residues 110 to 133, and helix α5 runs from residue 141 to residue 163. The topology of the bundle is 12354. There is a right-handed twist in the helical bundle, and helices α2 and α5 contain kinks. The interactions between helices α1 and α2 are predominantly hydrophobic, whereas the interactions between helices α2 and α3 are predominantly ionic. A combination of hydrophobic and polar interactions are formed between helices α3 and α5. Mostly polar interactions are found between helices α5 and α4, and hydrophobic interactions are formed between helices α4 and α1.Fig. 3Structure of ATP:cobalamin adenosyltransferase. A, ribbon diagram of one subunit of ATP:cobalamin adenosyltransferase. The monomer is composed of a single domain with a five-helix bundle core. The secondary structure elements are labeled α1-α5. B, ribbon diagram of trimeric ATP:cobalamin adenosyltransferase. The programs MOLSCRIPT and RASTER 3D were used in the production of the figures.View Large Image Figure ViewerDownload (PPT)The Oligomeric Structure—ATP:cobalamin adenosyltransferase was shown to be trimeric (Fig. 3B) using gel filtration studies (data not shown). This is consistent with analysis of the crystal structure. The oligomer interface is formed by the interaction of helix α2 from one subunit with helix α4 of another subunit and is stabilized by a combination of ionic and hydrophobic interactions. The ionic interactions that are formed between subunits A and B consist of the following: OE1 of Glu-27A and NE of Arg-127B; OE1 of Glu-27A and NH1 of Arg-128B; and OE2 of Glu-34A and NH1 of Arg-124B. These interactions are repeated between subunits B and C as well as between subunits C and A. Other ionic interactions at the interface are mediated through water molecules. A hydrophobic patch is formed between residues Phe-56A, Phe-63A, Val-105B and Trp-171B. This patch is also found between subunits B and C and between C and A.Active Site—The putative active site in ATP:cobalamin adenosyltransferase was identified by examining the location of the conserved residues. Twenty-one of the twenty-two highly conserved residues are located at the interface of two subunits but are not involved in subunit packing, suggesting a conserved function. Mutations in two of these residues are found in the MMAB gene in patients with the cblB disease of cobalamin metabolism, further supporting a key role for these residues. This region is formed at the interface of two subunits and is made up of helices α1 and α2 from one subunit and helices α3, α4, and α5 from the other subunit. The electrostatic surface representation of ATP:cobalamin adenosyltransferase shows that this same region is positively charged, which would allow the negatively charged ATP residue to be directed toward this area (Fig. 4). The disordered loops (residues 1-22 and 171-177) also map to this location of the protein. The active site in the apo-form of another ATP:cobalamin adenosyltransferase, CobA, was also disordered but became clearly defined in the presence of ligands (17Bauer C.B. Fonseca M.V. Holden H.M. Thoden J.B. Thompson T.B. Escalante-Semerena J.C. Rayment I. Biochemistry. 2001; 40: 361-374Crossref PubMed Scopus (62) Google Scholar). The residues important for ATP and HOCbl binding remain to be determined upon co-crystallization with these substrates.Fig. 4Electrostatic potential of ATP:cobalamin adenosyltransferase. Electrostatic potential ranges from -75 kiloteslas (red) to +42 kiloteslas (blue). The putative active site is positively charged and can attract negatively charged ATP molecules. SPOCK was used in the production of this figure.View Large Image Figure ViewerDownload (PPT)Structural Homologues—A number of structural homologues of ATP:cobalamin adenosyltransferase were identified using DALI (with Z-scores ranging from 7.4 to 9.9). The list of homologues includes rubrerythrin (1RYT; Z-score 9.9, 2.0 Å r.m.s.d.), bacterioferritin (1BCF; Z-score 9.5, 2.9 Å r.m.s.d.), non-heme iron-containing ferritin (1QGH; Z-score 9.2, 2.4 Å r.m.s.d.), PexB (1DPS; Z-score 9.2, 2.3 Å r.m.s.d.), and ferritin (1EUM; Z-score 7.6, 2.6 Å r.m.s.d.). The homologues belong to the ferritin superfamily, whose members have a four-helix bundle core structure (18Andrews S.C. Adv. Microb. Physiol. 1998; 40: 281-351Crossref PubMed Google Scholar). All ferritin superfamily members exist as oligomers of 12 or 24 similar subunits comprising the four-helix bundle core structure (19Ilari A. Stefanini S. Chiancone E. Tsernoglou D. Nat. Struct. Biol. 2000; 7: 38-43Crossref PubMed Scopus (226) Google Scholar). In the case of the dodecamer oligomeric structures (1QGH and 1DPS), they are composed of trimers placed at the four corners of a tetrahedron. Four of the five helices of ATP:cobalamin adenosyltransferase overlap with those found in the subunits of the five ferritin superfamily proteins; however, the length of the helices as well as the topology differ. Although the structural alignments have high significance, we believe that the homology is not related to function. Although ATP:cobalamin adenosyltransferase also forms trimers, as do some of the ferritin oligomers, it does not contain any of the conserved iron-binding residues and, therefore, is unlikely to function as an iron-binding protein. Also, the location of the iron-binding site in the ferritin superfamily is almost always found in the interior of the four-helix bundle, whereas the putative active site of ATP:cobalamin adenosyltransferase is located on the exterior of the protein at the interface of two subunits.Structure Comparison with CobA—Based on amino acid sequence analysis, there are three families of cobalamin adenosyltransferases, namely the CobA-type, the PduO-type, and the EutT-type (2Dobson C.M. Way T. Lecher D. Nadir H. Nearing M. Lerner-Ellis J.P. Hudson T.J. Rosenblatt D.S. Gravel R.A. Hum. Mol. Genet. 2002; 11: 3361-3369Crossref PubMed Scopus (134) Google Scholar). The recently determined crystal structure of CobA revealed a mixed α/β fold (17Bauer C.B. Fonseca M.V. Holden H.M. Thoden J.B. Thompson T.B. Escalante-Semerena J.C. Rayment I. Biochemistry. 2001; 40: 361-374Crossref PubMed Scopus (62) Google Scholar). By contrast, ATP:cobalamin adenosyltransferase contains a five-helix bundle. Thus, although CobA and ATP:cobalamin adenosyltransferase are both adenosyltransferases that generate AdoCbl, they have completely different structures. CobA and ATP:cobalamin adenosyltransferase also utilize different mechanisms for nucleotide binding and hydrolysis. CobA belongs to the P-loop-containing family of nucleotide hydrolases (17Bauer C.B. Fonseca M.V. Holden H.M. Thoden J.B. Thompson T.B. Escalante-Semerena J.C. Rayment I. Biochemistry. 2001; 40: 361-374Crossref PubMed Scopus (62) Google Scholar), whereas ATP: cobalamin adenosyltransferase is a PduO-type adenosyltransferase and, like PduO, has no recognizable P-loop for binding ATP. These differences in structure may contribute to the specificities of the adenosyltransferases for particular AdoCbl-d
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