Title: How the MccB bacterial ancestor of ubiquitin E1 initiates biosynthesis of the microcin C7 antibiotic
Abstract: Article4 June 2009free access How the MccB bacterial ancestor of ubiquitin E1 initiates biosynthesis of the microcin C7 antibiotic Catherine A Regni Catherine A Regni Department of Structural Biology, St Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Rebecca F Roush Rebecca F Roush Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Darcie J Miller Darcie J Miller Department of Structural Biology, St Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Amanda Nourse Amanda Nourse Hartwell Center for Bioinformatics and Biotechnology, St Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Christopher T Walsh Christopher T Walsh Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Brenda A Schulman Corresponding Author Brenda A Schulman Department of Structural Biology, St Jude Children's Research Hospital, Memphis, TN, USA Howard Hughes Medical Institute Search for more papers by this author Catherine A Regni Catherine A Regni Department of Structural Biology, St Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Rebecca F Roush Rebecca F Roush Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Darcie J Miller Darcie J Miller Department of Structural Biology, St Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Amanda Nourse Amanda Nourse Hartwell Center for Bioinformatics and Biotechnology, St Jude Children's Research Hospital, Memphis, TN, USA Search for more papers by this author Christopher T Walsh Christopher T Walsh Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Brenda A Schulman Corresponding Author Brenda A Schulman Department of Structural Biology, St Jude Children's Research Hospital, Memphis, TN, USA Howard Hughes Medical Institute Search for more papers by this author Author Information Catherine A Regni1, Rebecca F Roush2, Darcie J Miller1, Amanda Nourse3, Christopher T Walsh2 and Brenda A Schulman 1,4 1Department of Structural Biology, St Jude Children's Research Hospital, Memphis, TN, USA 2Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA 3Hartwell Center for Bioinformatics and Biotechnology, St Jude Children's Research Hospital, Memphis, TN, USA 4Howard Hughes Medical Institute *Corresponding author. Department of Structural Biology, St Jude Children's Research Hospital, MS #311, Room D5024E, 262 Danny Thomas Place, Memphis TN 38105, USA. Tel.: +1 901 595 5147; Fax: +1 901 595 5785; E-mail: [email protected] The EMBO Journal (2009)28:1953-1964https://doi.org/10.1038/emboj.2009.146 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The 39-kDa Escherichia coli enzyme MccB catalyses a remarkable posttranslational modification of the MccA heptapeptide during the biosynthesis of microcin C7 (MccC7), a 'Trojan horse' antibiotic. The approximately 260-residue C-terminal region of MccB is homologous to ubiquitin-like protein (UBL) activating enzyme (E1) adenylation domains. Accordingly, MccB-catalysed C-terminal MccA-acyl-adenylation is reminiscent of the E1-catalysed activation reaction. However, unlike E1 substrates, which are UBLs with a C-terminal di-glycine sequence, MccB's substrate, MccA, is a short peptide with an essential C-terminal Asn. Furthermore, after an intramolecular rearrangement of MccA-acyl-adenylate, MccB catalyses a second, unique reaction, producing a stable phosphoramidate-linked analogue of acyl-adenylated aspartic acid. We report six-crystal structures of MccB in apo, substrate-, intermediate-, and inhibitor-bound forms. Structural and kinetic analyses reveal a novel-peptide clamping mechanism for MccB binding to heptapeptide substrates and a dynamic-active site for catalysing dual adenosine triphosphate-consuming reactions. The results provide insight into how a distinctive member of the E1 superfamily carries out two-step activation for generating the peptidyl-antibiotic MccC7. Introduction To survive in a competitive milieu, bacteria generate a variety of antimicrobial agents that are toxic to rival species. Many clinically important antibiotics, such as penicillin, actinomycin, and vancomycin, contain peptide scaffolds that are generated by bacterial non-ribosomal peptide synthetases (Mootz et al, 2002). Other antibiotics contain ribosomally synthesized peptides, including the low-molecular weight microcins (Nolan and Walsh, 2008). Microcins are posttranslationally modified peptides that inhibit growth of competing Gram-negative bacteria, such as Escherichia, Salmonella, and Enterobacter, at nanomolar concentrations (Duquesne et al, 2007). Microcin C7 (MccC7, Figure 1A), a heptapeptide with an N-formylated methionine, an aminopropyl moiety, and a C-terminal phosphoramidate linkage to adenosine (Guijarro et al, 1995), is produced by Escherichia coli to eradicate competitor strains through a 'Trojan horse' mechanism. After import into target cells, the peptide framework of MccC7 is cleaved by non-specific peptidases to release a toxic adenylated-aspartic-acid mimic that targets aspartyl-tRNA synthetase, and thereby inhibits protein synthesis (Metlitskaya et al, 2006). Figure 1.The MccB reaction. (A) Structure of MccC7. (B) Mechanism for the MccB reaction. Only the C-terminus of the MccA heptapeptide is shown for clarity. (C) List of MccB structures presented. (D) Chemical structures of the peptides used in this study. Download figure Download PowerPoint Understanding the biosynthetic pathways of natural products, such as MccC7, allows us to gain insights into enzyme mechanism, provides opportunities for developing new antibiotics, and gives new prospects for protein design aimed at generating novel biologically active compounds. MccC7 is generated by two posttranslational modifications of a precursor ribosomal heptapeptide, MccA, featuring the sequence MRTGNAN. Posttranslational steps in MccC7 biosynthesis involve the conversion of the C-terminal Asn7 to an Asp amide, in which the nitrogen is linked by a phosphoramidate bond to adenosine monophosphate (AMP), followed by aminopropylation of one of the phosphate oxygens. The migration of the Asn7 carboxamido nitrogen and the N–P bond-forming step are catalysed in a Mg2+-dependent manner by the enzyme MccB (Figure 1B) (Roush et al, 2008). First, in a conventional acyl-adenylation reaction that consumes one adenosine triphosphate (ATP) molecule, MccB catalyses adenylation of the C-terminus of MccA. The resulting MccA-acyl-adenylate then undergoes intramolecular rearrangement, during which AMP is expelled by the carboxamido nitrogen of Asn7 as a peptidyl-succinimide intermediate is formed. Next, MccB catalyses an unusual adenylation of the succinimide: the succinimidyl nitrogen attacks the α-phosphate of a second ATP molecule. This links the AMP moiety to the peptide terminus through an N–P bond. The succinimide ring is hydrolysed by regiospecific water-mediated opening to yield the peptidyl-acyl-N-P-adenosine group, which is the Trojan horse reagent. Unlike acyl-adenylate complexes, which are hydrolytically labile-mixed acyl-phosphoric anhydrides, the phosphoramidate-linked analogue of aspartic acyl-adenylate (Figure 1B) is hydrolytically stable, allowing persistence throughout the remainder of the MccC7 biosynthetic pathway (aminopropylation, export by the producing cell and import by a sensitive bacterial cell) and ultimate toxic inhibition of aspartyl-tRNA synthetase and protein synthesis in competing bacteria. The 351-residue MccB has two discernible regions of sequence. The N-terminal (∼90 residues) region is not detectably homologous to known structures. The C-terminal (∼260 residues) region shares homology with the adenylation domain portion of ubiquitin-like (UBL) protein-activating enzymes, also called E1s, which initiate UBL conjugation (Supplementary Figures 1 and 2). MccB-catalysed adenylation of the MccA C-terminus is reminiscent of the first E1-catalysed reaction, which is the C-terminal adenylation of UBL substrates. However, the substrates of MccB and E1s are markedly divergent. Although MccB catalyses C-terminal transformation of a peptide destined for secretion to combat other organisms, substrates of E1s are proteins that serve as sulphur carriers during biosynthetic pathways, or as covalent protein modifiers (Hochstrasser, 2000, 2009). The sequence similarity between MccB and UBL-activating enzymes raises several questions about common and divergent aspects of the MccB mechanism. First, how does MccB recognize a short-peptide substrate, when all other family members have a ⩾8 kDa UBL protein substrate? Second, why does MccB catalyse adenylation of a peptide harbouring a C-terminal Asn or succinimide, rather than the Gly-Gly sequence found at the C-termini of UBLs? Third, how can MccB catalyse two successive adenylation reactions? To address these questions, we performed structural, biophysical, and biochemical analyses of MccB–MccA–nucleotide interactions. Results and discussion Overall structure of MccB We determined crystal structures of MccB separately and in complex with the following ligands: (1) MccA (first-peptide substrate), (2) MgATP (nucleotide substrate and divalent metal required for catalysis), (3) MccA and α,β-methylene ATP (AMPCPP) (first-peptide substrate and non-hydrolysable ATP analogue), (4) MccA–succinimide (second-peptide substrate), and (5) MccA–N7isoAsn (inhibitor) (Roush et al, 2008) (Figure 1C and D; Table I; Supplementary Figure 3). All asymmetric units contain two nearly identical MccB homodimers, with chains designated as A–B or C–D. Thus, the six structures presented here contain a total of 12 homodimers and 24 protomers. The 12 A–B and C–D homodimers superimpose with a Cα root mean square deviation (RMSD) of 76 Å. Owing to this structural similarity, our discussion focuses on chains A–B. The MccB homodimer has an elongated shape. Each homodimer has two parallel, two-domain 'active units'. Each 'active unit' contains an adenylation domain and a globular domain not found in other E1 enzymes. These domains are generated from parts of both protomers in the homodimer (Figure 2). Figure 2.Overall structure of MccB. The MccB–succinimide complex structure is shown, with MgATP modelled into the adenlyation-active site after superposition of MccB–succinimide and MccB–MgATP structures. The two protomers in the MccB homodimer are shown in cartoon representation in pink (chain A) and magenta (chain B), with their amino- and carboxyl-termini labelled N and C, respectively. Dashed lines represent connections between sequences that are not visible in the electron density. The MccB peptide clamp and adenylation domains are indicated with labels coloured to indicate chain A (pink) or B (magenta). MccA–succinimide (succinimide) and ATP are depicted as sticks, with their carbon atoms in yellow and grey, respectively. Nitrogen atoms are shown in blue, oxygen atoms in red, and phosphorous atoms in orange. Mg2+ and Zn2+ ions are depicted as cyan and grey spheres, respectively. Download figure Download PowerPoint Table 1. Data collection and refinement statistics Structure Apo MgATP MccA-N7isoAsn Succinimide MccA MccA-AMPCPP Space group P212121 P21 P21 P21 P21 P21 Data collection Native Inflection Remote λ (Å) 1.28290 1.28330 1.25000 0.97926 0.97926 1.00000 0.97926 0.97926 Unit cell a=144.7 a=144.5 a=144.8 a=55.9 a=55.9 a=55.9 a=56.3 a=55.8 b=145.0 b=144.7 b=144.9 b=138.2 b=138.2 b=138.0 b=138.6 b=138.0 c=158.7 c=158.1 c=158.9 c=80.2 c=80.8 c=80.1 c=81.4 c=80.8 α,β,γ=90 α,β,γ=90 α,β,γ=90 α,γ=90, β=92.6 α,γ=90, β=92.4 α,γ=90, β=92.1 α,γ=90, β=92.1 α,γ=90, β=92.2 Resolution 50–2.8 50–2.8 50–2.9 50–1.85 50–2.2 50–2.1 20–2.63 50–2.3 Rsyma (%) 10.1 (37.8) 8.6 (37.1) 10.9 (59.3) 5.4 (36.4) 6.5 (37.1) 9.0 (54.6) 8.3 (31.1) 8.1 (36.4) Completeness % 100 (99.8) 100(100) 100 (99.9) 92.9 (89.0) 96.7 (90.5) 99.9 (99.9) 100 (99.9) 98.9 (91.1) Redundancy 7.0 (6.5) 7.2 (7.1) 7.2 (7.1) 3.1 (2.8) 4.1 (3.6) 4.4 (4.2) 7.0 (6.4) 4.1 (3.3) Average I/σI 27.2 (4.5) 27.2 (4.6) 21.8 (2.6) 19.2 (2.7) 21.9 (3.0) 22.2 (2.2) 23.3 (4.5) 17.1 (2.1) Refinement Resolution 49.7–2.8 42.6–1.9 46.9–2.2 27.4–2.1 20–2.6 39.9–2.3 Reflections 77 699 84 354 57 050 67 014 36 931 51 038 Rworkb (%) 22.3 20.4 20.3 19.4 20.7 19.6 Rfreec (%) 25.7 25.6 26.1 25.0 27.3 25.5 Total atoms in refinement 11 064 11 310 11 321 10 714 10 571 10 853 Number of atoms Protein 11 049 10 425 10 577 10 319 10 298 10 412 Solvent 0 748 511 263 141 223 Peptide 0 — 194 118 118 76 Nucleotide 0 124 0 0 0 133 Zn2+, Mg2+, SO42− 4, 0, 0 4, 4, 1 4, 0, 7 4, 0, 2 4, 0, 2 4, 0, 1 Other 11 — — — — — RMSD bond (Å) 0.013 0.010 0.009 0.014 0.008 0.010 RMSD angle (°) 1.520 1.306 1.103 1.518 1.369 1.248 Average B (Å2) 53.2 22.9 36.3 45.1 52.0 44.2 Average B (Å2) Protein 53.9 22.5 36.0 44.8 51.5 43.7 Peptide — — 47.8 75.9 106.9 88.2 Nucleotide — 24.9 — — — 74.3 Solvent — 27.8 37.0 44.0 42.8 37.1 Zn2+, Mg2+, SO42− —, —, — 27.7, 32.4, 46.1 44.6, —, 67.3 51.2, —, 73.9 80.8, —, 96.7 56.3, —, 95.8 Other 59.4 — — — — — a ∑∣I−〈I〉∣/∑I, where I, observed intensity; 〈I〉, average intensity obtained from multiple observations of symmetry-related reflections. b Rwork=∑(∥Fp(obs)−Fp(calc)∥)/∑∣Fp(obs)∣. c Rfree=R factor for a selected subset (5%) of the reflections that was not included before refinement calculations. For the adenylation domain, the two parallel halves comprise residues (1) A104-A260, A287-A351, B90-B103 and (2) B104-B260, B287-B351, A90-A103. Each half includes a Rossmann-fold ATP-binding cleft. A five-stranded β-sheet, stabilized by an adjacent zinc-binding site, forms a concave peptide-binding surface. Both halves, individually and together, superimpose with the homologous regions of the bacterial UBL-activating enzymes MoeB and ThiF with Cα RMSDs of 1.5 Å over half and 1.7 Å over the dimer (Lake et al, 2001; Duda et al, 2005), and with the eukaryotic E1s UBA1, NAE1-UBA3, and SAE1-UBA2 with Cα RMSDs of 1.8–1.9 Å over half and 2.1 Å over the dimer (Walden et al, 2003b; Lois and Lima, 2005; Lee and Schindelin, 2008). The unique MccB domain is formed by the coupling of two separated regions of the MccB sequence: the N-terminal residues 1–87 from one monomer and residues 261–287 from the opposite protomer within the homodimeric assembly (Supplementary Figure 1). This latter region corresponds to an E1 enzyme 'crossover loop', which crosses over the C-terminal tail from the associated UBL (Lake et al, 2001; Walden et al, 2003a; Lois and Lima, 2005; Lee and Schindelin, 2008). In MccB, the 'crossover loop' is incorporated into the MccB-specific domain, which adopts a structure loosely related to a winged-helix fold. In our peptide co-crystal structures, numerous contacts are observed between this domain and the associated peptides. Thus, we refer to this domain as the 'peptide clamp', owing to its interactions with MccA. The adenylation and peptide clamp domains pack together in a manner resembling a clamshell. In the overall assembly of an 'active unit', each domain would represent half the shell, which together generate a large central groove that cradles the ATP and peptide substrates (Figure 2). Anchoring MccA through the unique peptide clamp Detailed insights into heptapeptide recognition come from superimposing the MccB–MccA and MccB–succinimide structures. MccA extends over MccB's adenylation domain β-sheet with its C-terminus approaching the ATP-binding site for adenylation (Figure 3A). The peptides bind in a groove formed by the adenylation domain β-sheet cleft on one side, and the peptide clamp on the other, burying an average of 940 A2 from MccA (85% of the total surface area) and ∼ 600 A2 from MccB. Figure 3.The unique MccB peptide clamp domain required for activity. (A) Close-up view of the MccB-binding site for peptide substrates. Structural superposition of MccB–MccA, (only MccA shown), MccB–succinimide, and MccB–MgATP (only MgATP shown). The two protomers in the MccB homodimer are shown in cartoon representation in pink and magenta. MccA, the succinimide intermediate, and ATP are depicted as sticks, with their carbon atoms in green, yellow, and grey, respectively. Nitrogen atoms are shown in blue, oxygen atoms in red, and phosphorous atoms in orange. The Mg2+ ion is shown as a cyan sphere. The MccB peptide clamp domain and associated peptide substrates are located inside the dotted box. (B) The separately expressed NTE of MccB (NTE) rescues the activity of truncation mutants lacking the NTE (ΔNTE). HPLC traces (220 nm) showing the product resulting from incubation of MccA with MccB ΔNTE (10 μM), isolated NTE (100 μM), or both in reaction buffer for 2 h at room temperature. Download figure Download PowerPoint To test the functional importance of the peptide clamp domain, we performed enzymatic assays on mutant versions of MccB. Deleting the MccB N-terminal extension (ΔNTE), which comprises the bulk of the peptide clamp domain, completely eliminates the activity (Figure 3B; Supplementary Figure 4). To test whether a ΔNTE mutant has an intact adenylation domain assembly, we assayed homodimerization by analytical ultracentrifugation (AUC). As the AUC data indicate dimerization, reflecting proper folding (Supplementary Figure 5), we tested whether adding a 10-kDa protein fragment corresponding to MccB's N-terminal 87 residues (NTE) could restore any activity to the ΔNTE mutant in trans. Approximately 20% of the wild-type (WT) activity was restored (Figure 3B; Table II). These results support the importance of the peptide clamp domain, and indicate that the adenylation and peptide clamp domains work together optimally when expressed as a single unit. Table 2. MccB mutants kinetics Mutant MccA ATP Km (μM) kcat (h−1) kcat/Km (h−1 μM−1) Km (μM) kcat (h−1) kcat/Km (h−1 μM−1) MccB (WT) (Roush et al, 2008) 61±16 6.4±1.8 0.1 70±30 12.5±2.4 0.18 MccB K10A 370±240 2.8±1.2 0.007 120±20 3.7±0.5 0.03 MccB R94A 950±260 2.8±1.9 0.003 380±130 4.5±3.3 0.008 MccB C123V 60±15 6.0±1.5 0.1 270±140 7.0±2.9 0.02 MccB C123L 49±10 1.8±0.3 0.04 220±80 1.1±0.3 0.008 MccB D214A —Not active— —Not active— MccB H215A 100±40 5.6±2.3 0.05 150±40 7.5±2.6 0.05 MccB Y239A 250±70 0.01±0.006 0.00003 380±190 0.02±0.008 0.00005 MccB N241A 440±140 0.8±0.4 0.002 400±120 1.7±0.5 0.004 MccB ΔNTE+NTE 700±200 1.1±0.4 0.002 3800±1900 2.9±1.2 0.0007 MccB ΔNTE —Not active— —Not active— MccB NTE —Not active— —Not active— Certain MccB residues from both the clamp and adenylation domains contact the peptides in all the co-crystal structures (Figure 4). Two such residues from the clamp domain are K10 and E26. MccB's K10 directly contacts the carbonyl oxygen from G4 and the T3 side chain from MccA and the succinimide. This latter interaction may have a function in constraining the sequences allowed at MccA position 3 during MccC7 biosynthesis (Kazakov et al, 2007). The MccB–K10A mutant has little effect on catalytic rate, but has an increase in the Km of MccA (Table II), which further shows its importance for peptide binding. Figure 4.MccB binding to MccA. Stereo panels showing contacts between MccB and the MccA peptide ligands. The two protomers in the MccB homodimer are shown in cartoon representation in pink and magenta. MccB side chains and peptides are depicted as sticks, and contacts within 5 Å are indicated by dashes. (A) MccA, green. (B) MccA–succinimide, yellow. (C) MccA–N7isoAsn, orange. Nitrogen atoms are shown in blue and oxygens in red. Download figure Download PowerPoint Residues in the adenylation domain form a pocket, in which the N-terminus of the peptides binds. The peptide N-terminal Met1 adopts a similar conformation in all the complexes, and is encircled by a deep hydrophobic channel generated by residues I243, V245, W326, and H333. The carbonyl of the peptide Met1 participates in a hydrogen bonding network with R322 and Q335 (Figure 4). In the absence of peptide (as in the ATP-bound structure), R322 and Q335 are bound to the solvent. Past Met1, the next four peptide residues follow related trajectories towards the adenylation-active site. Although none of these structurally represents a catalytic intermediate, the C-terminus of the succinimide peptide comes close to approaching the adenylation-active site. In the crystals, the C-terminus of the peptide substrate MccA occupies several locations, none of which is as close to the adenylation-active site as the succinimide. A different backbone conformation is observed for the MccA–N7isoAsn inhibitor, which is a subtle variant of the first-peptide substrate. Residues Thr3-Asn5 of the inhibitor are 'looped out' by one residue relative to the corresponding substrate structures, such that the Ala6-isoAsn7 backbone aligns with Asn5-Ala6 from the succinimide peptide. Thus, the C-terminus of MccA–N7isoAsn does not come as close to the adenylation-active site as the succinimide peptide. The conformational variability observed at the C-termini of the peptides will be discussed below. Comparison of the peptide-MccB structures with UBLs complexed with their activating enzymes (Lake et al, 2001; Walden et al, 2003a; Lois and Lima, 2005; Lehmann et al, 2006; Huang et al, 2007; Lee and Schindelin, 2008) reveals that the MccA-derived peptides resemble the last seven residues of E1-bound UBLs (Supplementary Figure 6). Thus, it seems that the unique MccB peptide clamping mechanisms direct substrates to an E1-like active site. Adenylation-active site ATP binds in a cleft centred around the MccB Gly-Cys-Gly-Gly-Ile-Gly nucleotide-binding motif, near the C-terminal end of the MccA-derived peptides. Many features of MccB's ATP-binding site resemble those of MoeB/ThiF and E1s, with both protomers in the MccB dimer contributing to the ATP-binding site (Figure 5A; Supplementary Figure 7) (Lake et al, 2001; Walden et al, 2003a; Duda et al, 2005; Lois and Lima, 2005; Huang et al, 2007). A hydrophobic pocket (L121, I194, A213, L219) binds ATP's adenine. Polar residues (N154, R157, Q158, K170) contact ATP's ribose and phosphates. An Arg finger (94) is provided by the opposite monomer in the complex. Mutation of the Arg finger to alanine (R94A) has no significant effect on the kcat, but increases the Km for both MccA and ATP (Table II). Figure 5.A common adenylation-active site for MccA and MccA–succinimide. (A) Close-up views of the MccB adenylation-active site is shown in stereo. The MccB–MgATP structure is shown, with the succinimide intermediate modelled after structural superposition of MccB–MgATP and MccB–succinimide complexes. MccB is depicted as in Figure 2 with side chains shown as sticks, and contacts within 5 Å indicated by dashes. (B) MccB D214A is unable to catalyse generation of the succinimide intermediate or product from MccA. HPLC traces (220 and 260 nm) showing lack of product formation after incubation of MccA and ATP with MccB D214A in reaction buffer for 5 h at room temperature. Further incubation to 24 h gave no product. (C) The MccB Y239A mutation significantly hinders enzymatic activity for reactions starting with MccA. HPLC traces (220 and 260 nm) showing the product resulting from incubation of MccA with MccB–Y239A (10.5 μM) in reaction buffer for 24 h at room temperature. (D) MccB D214A and Y239A are defective at converting the succinimide to product. HPLC traces (220 nm) showing incubations of WT MccB (100 nM), MccB–D214A (15 μM), or MccB–Y239A (15 μM) mutants with 100 μM MccA–succinimide in reaction buffer for 3.5 h at room temperature. Download figure Download PowerPoint Mg2+ is coordinated by the three ATP phosphates and D214. D214 is absolutely conserved as an Asp in all UBL-activating enzymes (Supplementary Figure 2). The corresponding Asp residue was shown earlier to be essential for catalysis by the UBL-activating enzymes E. coli MoeB and the human NEDD8 E1 (Lake et al, 2001; Walden et al, 2003b). Furthermore, mutation of the corresponding Asp in the ubiquitin E1, UBA1, had pleiotropic effects, including increasing the Km for ATP and ubiquitin and decreasing the kcat for many steps in ubiquitination (Tokgoz et al, 2006). The earlier studies on UBA1 support a dual function of the Asp in binding MgATP and transition state stabilization (Tokgoz et al, 2006). The MccB residue D214 is essential for catalysis; we were unable to detect conversion of MccA to succinimide or product by the D214A mutant. (Figure 5B). However, coincubation of the D214A and ΔNTE mutants with MccA yields activity similar to mixing the isolated NTE with the ΔNTE mutant (Supplementary Figure 8). The former result could either be explained by complementation in trans or by a low concentration of mixed dimers in which ΔNTE would contribute the catalytic D214, and the full-length version harbouring the D214A mutation would contribute a peptide clamp to form one complete-active site per heterodimer. In either case, the finding of limited activity indicates the importance of the adenylation-active site working in concert with the peptide clamp domain. Unique features of the MccB-active site In addition to the shared properties with E1s, the MccB Y239 stands out as being unique in the adenylation catalytic site. Y239 contacts both the ATP α-phosphate and the heptapeptide substrate C-terminus (Figure 5A). Y239 is located on the opposite side of MccA from the Mg2+-coordinating D214. Together, Y239 and D214 seem to form a bridge. The MccA and succinimide peptide substrates would need to pass into a channel under the D214–Y239 bridge for adenylation. Y239 has an important catalytic role, as illustrated by the ∼five-fold increase in Km for both ATP and MccA and >600-fold decrease in kcat in the Y239A mutant (Table II; Figure 5C). Notably, the MccA C-terminus is atypical among substrates of UBL-activating enzymes as not terminating in the sequence Gly-Gly. Structural comparison reveals that UBL penultimate Glys are oriented by a key E1 Arg, which is lacking and substituted with I220 in MccB. MccB's aforementioned unique Y239 may also prevent Gly-Gly recognition. The corresponding E1 residue is a small Ala or Thr, which allows proper orientation of a UBL C-terminal Gly-Gly. In contrast, the MccB adenylation-active site is slightly displaced as compared with UBL-activating enzymes, with Y239 altering the conformation of the MccA-contacting β-strand13, and obscuring the path taken by a UBL Gly-Gly (Supplementary Figure 9). The channel leading under the D214–Y239 bridge and towards ATP is lined with backbone hydroxyls, which may also favour binding to MccA's C-terminal Asn, and prevent binding of closely related peptides as observed for MccA-N7D (Roush et al, 2008). Successive adenylation reactions at a common-active site A striking feature of the MccB mechanism is the usage of two ATP molecules per catalytic cycle: one for the adenylation of MccA and the second for the formation of the N–P bond through the adenylation of the succinimide intermediate (Roush et al, 2008). The mutants D214A and Y239A have significant reductions in overall turnover (Figure 5B and C); we were unable to detect any product formation by D214A starting from the heptapeptide substrate MccA (Figure 5B). The structural data suggests that there is one common-active site for MccA and the succinimide intermediate, so we therefore evaluated the processing for the synthetic peptidyl-succinimide intermediate on the second half reaction with our most deleterious point mutants, D214A and Y239A. The detection limit for high-performance liquid chromatography (HPLC) assays was 10 pmol of MccB product, which amounted to a rate of 0.01% of WT enzyme over a 24-h time period. The conversion of the succinimide intermediate to product, representing the second ATP consumption step, occurs 40 times faster than the overall reaction starting with MccA (Roush et al, 2008). Therefore, assaying the mutants with the succinimide intermediate is more sensitive. On incubation of WT MccB (0.1 μM), or the D214A or Y239A mutants at 150-fold higher enzyme concentrations (15 μM) to magnify sensitivity with 100 μM peptidyl-succinimide, product formation was detected (Figure 5D). D214A was ∼150-fold slower than WT, and Y239A was ∼600-fold slower than WT MccB in this second half reaction. Therefore, the D214A and Y239A mutations markedly affect both overall turnover and the peptidyl-succinimide to acyl-phosphoramidate product step. As the peptidyl-succinimide does not accumulate in assays of D214A and Y239A starting with MccA (data not shown), we intuit that both the first and the second ATP-using steps are slowed down more than two orders of magnitude and that the formation of the heptapeptidyl-AMP, the cyclization to the succinimide, and the rearrangement to the final N–P-linked product occur in the same active site. Conformational variability of MccB, MccA-derived heptapeptides, a