Characterization of NosI

ACPs typically function as molecular carriers. In fatty acid biosynthesis, they serve as the template upon which fatty acyl chains are elongated.²² In nonribosomal peptide synthetases (NRPSs) and polyketide synthetases (PKSs), ACPs exist as carrier or thiolation domains (TDs).²³ In PKSs, acyl-CoAs existing as primary metabolites are attached to TDs via transthioesterification reactions. In NRPSs, however, amino acids and other metabolites that do not exist naturally as CoA-thioesters must first be activated in an ATP-dependent fashion to give an intermediate amino acid–adenylate species, which is then used to generate the corresponding TD-amino acid thioester.²³ In the nos operon, NosI is annotated as requiring ATP (AMP-dependent acyl-CoA synthetase), suggesting that it might be involved in the activation and transfer of MIA to holo NosJ. The nosI gene was therefore cloned such that its product would be produced with an N-terminal hexahistidine (His₆) tag upon expression to facilitate purification by immobilized metal affinity chromatography (IMAC). The protein was purified to >90% homogeneity with a yield of ~30 mg from 30 g of E. coli cell paste (Figure S2). The addition of 5 mM ATP to purification buffers was critical for improving the solubility of the protein.

To assess whether NosI activates MIA, NosI was incubated with 1 mM MIA and 1 mM ATP in HEPES buffer, pH 7.5, and formation of the MIA-adenylate was assessed at designated times by ESI⁺-MS (Figure ​Figure44A). A burst phase of MIA-AMP production is observed that is followed by a second phase that is nearly horizontal, suggesting that no additional product formation, or extremely slow product formation, takes place after the initial burst. When the experiment is conducted with varying concentrations of NosI (2, 4, or 8 μM), the amplitudes of the burst phases vary linearly with those concentrations, suggesting that the enzyme catalyzes only one turnover under the conditions described (Figure ​Figure44A,B). This behavior suggests that NosI catalyzes both the activation of MIA and its subsequent transfer to some target.

Open in a separate window

Figure 4

Reaction of NosI with ATP and MIA. ATP (1 mM) and MIA (1 mM) were incubated with 2 (red triangles), 4 (black triangles), 8 μM (green triangles) NosI in the absence of NosK for varying lengths of time (15 to 800 s), and MIA-AMP was measured by LC-MS as a relative integrated response (A). Amplitudes from each of the time courses plotted as a function of NosI concentration show a linear correlation (B). The reactions were conducted in an ice–water bath.

To assess whether holo NosJ is the NosI-dependent acceptor of MIA, NosI was incubated with MIA, ATP, and NosJ, and formation of NosJ-MIA was assessed by HPLC. As shown in Figure ​Figure55 (Trace B), apo and holo NosJ migrate at retention times of 9.8 (black trace) and 10.6 min (blue trace), respectively, under the conditions described in the Experimental Procedures. When holo NosJ is incubated with NosI, ATP, and MIA, this retention time shifts to 19.3 min, indicating the modification of holo NosJ with MIA (Figure ​Figure55, Trace F). Omission of NosI (Trace C), ATP (Trace D), or MIA (Trace E) from the reaction mixture does not result in a shift, in the retention time of holo NosJ, verifying that all of these components are required for modification of holo NosJ. Analysis of NosJ by mass spectrometry after incubating it with NosI, MIA, and ATP revealed it to exhibit an m/z of 9215 (Figure ​Figure33, panel 3), which is consistent with the theoretical mass of MIA-NosJ (9215.13 Da).

Open in a separate window

Figure 5

HPLC analysis of holo NosJ upon reaction with ATP, MIA, and NosI. (A) apo NosJ; (B) holo NosJ; (C) holo NosJ + ATP and MIA; (D) holo NosJ + NosI and MIA; (E) holo NosJ + NosI and ATP; and (F) holo NosJ + NosI, MIA, and ATP (complete reaction).

Characterization of NosK

In the nos operon, nosJ is adjacent to nosK. In the noc operon, which encodes proteins that are involved in the biosynthesis of nocathiacin, a related thiopeptide antibiotic, the encoded NocJ and NocK proteins are part of the same polypeptide. These observations suggest that NosJ and NosK might have related activities. Therefore, hexahistidine-tagged NosK was purified to homogeneity (100 mg from 10 g of E. coli cell paste) by IMAC (Figure S3) and characterized. As shown in Figure ​Figure66 (blue trace), NosK migrates at a retention time of 7.6 min under the conditions described in the Experimental Procedures. The addition of holo NosJ, MIA, and ATP to NosK results in essentially no change in retention time (Figure ​Figure66 black trace). However, the addition of NosI to holo NosJ, MIA, ATP, and NosK results in a change in NosK’s retention time from 7.6 to 10 min. When NosK is then digested with trypsin and subjected to mass spectrometry, a peptide is observed (LIGAS₁₀₂YLGGPLAHR) that contains MIA covalently bound to the side-chain of Ser102 (Table S1). In a control reaction, in which ATP is omitted, the same peptide is observed but without the MIA linkage (Table S2).

Open in a separate window

Figure 6

HPLC analysis of the acylation of NosK. NosK, blue; NosK + NosJ, black; NosK + NosJ + NosI, pink; NosK Ser102Ala variant, red; NosK Ser102Ala variant + NosJ + NosI, green.

To confirm the role of Ser102 as an acyl acceptor, the amino acid was changed to alanine and the resulting NosK variant was substituted in the reaction. NosK S102A migrates slightly slower than wild-type NosK, exhibiting a retention time of 8.3 min rather than 7.7 min (Figure ​Figure66, red trace). When the NosK S102A variant is incubated with holo NosJ, ATP, MIA, and NosI, no shift in retention time is observed, which is consistent with Ser102 as the intermediate acyl acceptor site. This role for NosK is also consistent with observations reported in Figure ​Figure33 (panel 4), wherein the addition of NosK to a reaction containing NosI, NosJ, MIA, and ATP results in the conversion of MIA-NosJ to holo NosJ.

General Methods

DNA sequencing was carried out at the Pennsylvania State University Genomic Core facility. UV–visible spectra were recorded on a Cary 50 spectrometer from Varian (Walnut Creek, CA) using the WinUV software package to direct the instrument and manipulate the data. High-performance liquid chromatography (HPLC) was conducted on an Agilent 1100 Series system coupled with an Agilent 1100 Series variable wavelength detector and quaternary pump. Polymerase chain reactions (PCR) were performed using a robocylcer gradient 40 thermal cycler from Stratagene.

Cloning of the nosI Gene into pET-28a

The gene encoding nosI was codon-optimized for expression in E. coli and was delivered as a construct in plasmid pMK-RQ. The gene contained two restriction sites, NdeI (5′ end) and EcoRI (3′ end), and was cloned into the same restriction sites of a pET-28a vector. The nosI gene was expressed as a fusion protein with an N-terminal hexahistidine (His₆) tag. The codon-optimized gene sequence of nosI, with the NdeI and EcoRI restriction sites indicated in bold type, is provided in the Supporting Information.

Cloning of the nosJ Gene into pET-26b

The gene encoding nosJ was codon-optimized for expression in E. coli and was delivered as a construct in plasmid pMA-T. The gene contained two restriction sites, NdeI (5′ end) and EcoRI (3′ end), with a stop codon just before the EcoRI site. The gene was excised by digestion with NdeI and EcoRI and cloned into a pET-26b vector that was digested with the same two enzymes. The codon-optimized gene sequence of nosJ, with the NdeI and EcoRI restriction sites indicated in bold type, is provided in the Supporting Information.

Cloning of the nosK Gene into pET-28a

The gene encoding nosK was codon-optimized for expression in E. coli and was delivered as a construct in plasmid pMA-T. The gene contained two restriction sites, NdeI (5′ end) and EcoRI (3′ end), and was cloned into the same restriction sites of a pET-28a vector. The listed primers were used to introduce a stop codon at amino acid position 271. The nosK gene was expressed as a fusion protein with an N-terminal hexahistidine tag. The codon-optimized gene sequence of nosK, with the NdeI and EcoRI restriction sites indicated in bold type, is provided in the Supporting Information.

Construction of the NosK Ser102Ala Variant

The gene encoding the Ser102Ala variant was constructed by the polymerase chain reaction. The reaction contained 200 ng of template, 0.4 μM forward primer (5′-GGTCCGGGTCGTCTGATTGGTGCAGCGTATCTGGGTGGTCCGCTGGC), 0.4 μM reverse primer (5′-GCCAGCGGACCACCCAGATACGCTGCACCAATCAGACGACCCGGACC-3′), 250 μM each dNTP, 1× PfuTurbo DNA polymerase buffer, and 2.5 U of PfuTurbo DNA polymerase AD in a total of 50 μL. The reaction was heated at 95 °C for 5 min to denature the template. Next, 16 cycles of denaturation, annealing, and extension were performed at 95 °C for 1 min, 55 °C for 1.5 min, and 68 °C for 15 min, respectively. A final extension was performed at 68 °C for 10 min.

Overproduction and Purification of NosI

E. coli BL-21 (DE3) was transformed with plasmids encoding NosI. An LB media starter culture was inoculated with a single colony and incubated overnight (250 rpm at 37 °C). Several shake flasks of LB media were then inoculated with the overnight starter culture to initiate growth (180 rpm at 37 °C). Gene expression was induced with 0.5 mM IPTG at an OD₆₀₀ of ~0.6, and the flasks were subsequently placed in an ice bath for 1 h. Once cooled, the cultures were incubated overnight (approximately 18 h) at 18 °C with shaking at 180 rpm. The cells were harvested (~30 g), flash frozen in liquid nitrogen, and stored −80 °C.

For purification, 30 g of cell paste was resuspended in 150 mL of lysis buffer (50 mM HEPES, pH 7.5, 300 mM KCl, 10% glycerol, 5 mM ATP, and 10 mM BME) containing PMSF (1 mM), DNase1 (100 μg mL^–1), and lysozyme (1 mg mL^–1). Once the cells were resuspended, they were placed in an ice-bath and subjected to 6 sonication bursts (40% output) of 45 s with 8 min pauses between bursts to allow the temperature to re-equilibrate. The lysate was then centrifuged for 1 h at 50,000g at 4 °C. The resulting supernatant was loaded onto a Talon cobalt resin column already equilibrated with 100 mL of lysis buffer. The loaded column was washed twice with 100 mL of lysis buffer before eluting it with 50 mL of elution buffer (50 mM HEPES, pH 7.5, 300 mM KCl, 250 mM imidazole, 10% glycerol, 1 mM ATP, and 10 mM BME). Two 20 mL fractions were concentrated using an Amicon Ultra centrifugal filter unit (Millipore; Billerica, MA) with a 10-kDa molecular weight cutoff membrane. The protein was purified further by passing it over a 16/60 S-200 column equilibrated in 300 mM KCl, 50 mM HEPES, pH 7.5, 1 mM ATP, 1 mM DTT, and 20% glycerol. The protein was then concentrated in the same manner as described above. The concentration of protein was estimated by the Bradford assay.³² The protein was flash frozen in microcentrifuge tubes and stored at −80 °C. The total yield is ~15 mg from 30 g of frozen cell paste.

Overproduction and Purification of NosJ

Plasmids encoding NosJ were transformed into BL-21 (DE3). An LB media starter culture was then inoculated with a single colony and incubated overnight with shaking (250 rpm) at 37 °C. Several 6 L shake flasks of LB media were then inoculated with the overnight starter culture and agitated at 180 rpm and 37 °C. Gene expression was induced with 1 mM IPTG at an OD₆₀₀ of ~0.6, and the flasks were subsequently placed in an ice bath for 1 h. Once cooled, the cultures were incubated overnight (approximately 18 h) at 18 °C with shaking (180 rpm). The cells were harvested (~30 g), flash frozen in liquid nitrogen, and stored in a liquid nitrogen dewar until purification.

For purification, 30 g of cell paste was resuspended in 150 mL of lysis buffer (50 mM Tris–HCl, pH 8.4) containing DNase1 (100 μg mL^–1) and lysozyme (1 mg mL^–1). Once resuspended, the mixture was placed in an ice bath and sonicated (40% output) for 45 s with 7 min pauses between bursts, for a total sonication time of 6 min. The lysate was then centrifuged at 35,000g for 1 h at 4 °C. The final supernatant was then diluted with 1 volume of 50 mM MES, pH 6.1, and then loaded onto a DE-52 anion-exchange column equilibrated in 50 mM MES buffer, pH 6.1. Once loaded, the column was washed with 1 L of 50 mM MES, pH 6.1, and the protein was eluted with a linear 1 L gradient of 0 to 0.50 M KCl in 50 mM MES, pH 6.1. Fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and those containing significant amounts of NosJ (350 mL total volume) were pooled and concentrated in an Amicon stirred cell with a 3-kDa molecular weight cutoff membrane. The concentrated protein was further purified on a HiPrep 26/60 S-200 column equilibrated in 25 mM HEPES, pH 7.5, 10% glycerol, and 300 mM KCl. The protein was concentrated using an ultracentrifugal filter, flash frozen in liquid nitrogen, and stored in a liquid nitrogen dewar. The final concentration was determined using an extinction coefficient of 12 490 L mol^–1 cm^–1 mol at 280 nm, which was determined from the sequence of NosJ using the ProtParam tool from the ExPASy Proteomics Resource Portal (http://web.expasy.org/protparam/). The final yield was ~60 mg from 30 g of cell paste.

Overproduction and Purification of WT NosK and the NosK Ser102Ala Variant

Plasmids encoding WT NosK or the NosK Ser102Ala variant were transformed into E. coli BL-21 (DE3). An LB media starter culture was then inoculated with a single colony and incubated overnight at 37 °C with shaking at 250 rpm. Several shake flasks of LB media were then inoculated with the overnight starter culture to initiate growth (180 rpm at 37 °C). Gene expression was induced with 1 mM IPTG at an OD₆₀₀ of ~0.6, and the flasks were immediately placed in an ice bath for 1 h. Once cooled, the cultures were incubated overnight for 18 h at 18 °C with shaking at 180 rpm. The cells were harvested (~20 g), flash-frozen in liquid nitrogen, and stored at −80 °C.

For purification, 20 g of frozen cell paste was resuspended in 150 mL of lysis buffer (50 mM HEPES, pH 7.5, 300 mM KCl, 10% (v/v) glycerol, and 10 mM BME) containing PMSF (1 mM), DNase1 (100 μg mL^–1), and lysozyme (1 mg mL^–1). Resuspended cells were incubated on ice and subjected to six sonication bursts on a QSonica instrument (40% output) for 45 s each with 8 min intermittent pauses. The lysate was then centrifuged for 1 h at 50,000g at 4 °C. The supernatant was then loaded onto Talon Co(2⁺) resin equilibrated in the lysis buffer. The resin was washed twice with 100 mL of the lysis buffer prior to elution of His-tagged NosK with 50 mL of elution buffer (50 mM HEPES, pH 7.5, 30 mM KCl, 250 mM imidazole, 10% (v/v) glycerol, and 10 mM BME). The pooled eluate was concentrated by ultracentrifugation using an Amicon Ultra centrifugal filter unit with a 10-kDa molecular weight cutoff membrane. The protein was purified further by passage over a HiPrep 16/60 S200 column equilibrated in 300 mM KCl, 50 mM HEPES, pH 7.5, 1 mM DTT, and 10% (v/v) glycerol. The protein was then concentrated in this buffer as described above and stored in liquid nitrogen. The final concentration of NosK was estimated by UV–vis spectroscopy using an extinction coefficient of 29 450 L mol^–1 cm^–1 at 280 nm obtained using the ProtParam tool of the ExPASy proteomics resource portal (http://web.expasy.org/protparam/). The final yield was ~100 mg of protein per 20 g of cell paste.

NosI Activity Determinations

Reaction mixtures contained 25 mM HEPES, pH 7.5, 1 mM ATP, 1 mM MIA, and varying concentrations of NosI (2, 4, and 8 μM), and were performed in triplicate. All reaction components except MIA were mixed and then equilibrated in an ice–water bath in which the reactions were conducted. Reactions were initiated by the addition of MIA, and at designated times, 10 μL aliquots were removed and added to 10 mL of a solution containing 100 μM tryptophan (internal standard) and 50 mM H₂SO₄ to quench the reaction. Products were then analyzed by HPLC with detection by mass spectrometry (LC-MS).

LC-MS Method

HPLC with detection by positive-mode electrospray-ionization (ESI⁺) mass spectrometry was conducted on an Agilent Technologies 1260 system coupled to an Agilent Technologies 6460 triple-quad mass spectrometer. The associated MassHunter software package was used to operate the system and to collect and analyze the data. Each quenched reaction was injected onto an Agilent Technologies Zorbax Rapid Resolution XBD-C18 column (4.6 mm × 50 mm, 1.8 μM particle size). The column was first equilibrated in 95% solvent A (0.1% formic acid) and 5% solvent B (100% acetonitrile). From 0 to 2 min, a linear gradient from 5% to 60% solvent B was applied. From 2 to 2.5 min, the gradient was increased linearly from 60% to 100% solvent B. From 2.5 to 4.5 min, the gradient was decreased linearly to 5% solvent B and was then held constant for an additional 5 min. A constant flow rate of 0.7 mL/min was used throughout the method. MIA-AMP and tryptophan were detected using the single-ion monitoring (SIM) mode. The set fragmentor voltages for MIA-AMP and for tryptophan were 135 and 130 V, respectively.

Analysis of the Reaction Containing NosI, NosJ, and NosK

The basic reaction mixture consisted of 25 mM HEPES, pH 7.5, 1 mM CoA, and 400 μM NosJ in a total volume of 40 μL. For formation of holo NosJ, 36.7 μM Ec holo ACP synthase, prepared as previously described,^19,20 was included. For formation of MIA-holo NosJ, 20 μM NosI, 2 mM ATP, 2 mM MIA, 100 mM MgCl₂, and 36.7 μM Ec holo ACP synthase was included. Control reactions had either ATP, MIA, or NosI omitted. Assays were quenched with 0.2% TFA, pH 2.4, after 2 h at room temperature, and then analyzed by HPLC.

Samples were analyzed using the Agilent 1100 series HPLC ChemStation software. A Zorbax 300 stable bond C18 5 μm 4.6 mm × 250 mm column was used with three different solvents at a flow rate of 1.3 mL/min: solvent A, 0.2% TFA, pH 2.4; solvent B, acetonitrile; and solvent C, methanol. The column was equilibrated in 5% solvent B and 95% solvent A, which was maintained for 30 s after injection. From 30 s to 4 min, solvents B and C were increased to 39% and 9%, respectively. From 4 to 12 min, solvent C was increased to 11.8%, while solvent B was held constant at 39%. From 12 to 22 min, solvent C was increased to 50%, while solvent B was held constant at 39%. From 22 to 25 min, solvents B and C were held constant at 39% and 50%, respectively. From 25 to 27 min, solvent B was increased to 100%. From 27 to 28 min, solvent B was held constant at 100%. From 28 to 29.5 min, solvent B was decreased to 5%. From 29.5 to 32 min, solvent B was held constant at 5%.

MS Analysis of NosI, NosJ, and NosK Reactions

The complete reaction (Figure ​Figure33, panel 4) contained the following in a final volume of 400 μL: 300 μM NosJ, 18 μM holo ACP synthase, 25 μM NosI, 300 μM NosK, 25 mM HEPES, pH 7.5, 500 μM CoA, 1 mM ATP, 1 mM MIA, and 100 mM MgCl₂. For some reactions, various components of the complete reaction mixture were omitted as indicated in the text and legend for Figure ​Figure33. The reaction corresponding to panel 1 contained 300 μM NosJ, 25 mM HEPES, pH 7.5, 400 μM CoA, and 100 mM MgCl₂. The reaction corresponding to panel 2 contained 300 μM NosJ, 25 mM HEPES, pH 7.5, 1 mM CoA, 100 mM MgCl₂, and 18 μM Ec holo ACP synthase. The reaction corresponding to panel 3 contained 300 μM NosJ, 20 μM NosI, 25 mM HEPES, pH 7.5, 500 μM CoA, 100 mM MgCl₂, 1 mM ATP, 1 mM MIA, and 18 μM Ec holo ACP synthase. The reactions were incubated at room temperature for at least 1 h before being frozen and stored at −80 °C until ready for analysis.

MS analysis was performed on a Waters Q-TOF Premier quadrupole/time-of-flight (TOF) mass spectrometer (Waters Corporation, Micromass Ltd., Manchester, UK). Operation of the mass spectrometer was performed using MassLynx software Version 4.1 (http://www.waters.com). Samples were introduced into the mass spectrometer using a Waters 2695 HPLC system. Separation was performed by gradient elution on a Thermo BioBasic C4, 50 × 2.1 mm HPLC column. The mobile phases used were 0.1% formic acid in deionized water (solvent A), and 0.1% formic acid in acetonitrile (solvent B) at a flow rate of 0.2 mL/min. The column was equilibrated in 2% solvent B, which was held constant for 1 min after sample (15 μL) injection. From 1.0 to 2.0 min, a linear gradient from 2% to 95% solvent B was applied, which was held constant at 95% solvent B from 2.0 to 15.0 min. From 15.0 to 15.1 min, a linear gradient from 95% to 2% solvent B was applied, which was held constant at 2% solvent B from 15.1 to 20.0 min. Data acquisition was performed during the first 15 min of the run, whereas the final 5 min was used for column re-equilibration. The nitrogen drying gas temperature was set to 300 °C at a flow rate of 6 L/min. The capillary voltage was 2.8 kV. The mass spectrometer was set to scan from 500 to 2500 m/z in positive ion mode, using electrospray ionization.

MS Analysis of Trypsin-Digested NosK

The reaction mixture consisted of 50 μM NosJ, 50 μM NosI, 500 μM NosK, 100 mM MgCl₂, 2 mM ATP, 2 mM MIA, 25 mM HEPES, pH 7.5, 0.1 mM CoA, and 10 μM holo ACP synthase in a total volume of 30 μL. The reaction was incubated at room temperature for approximately 1 h and then quenched with 30 μL of 2× SDS-PAGE buffer. A control reaction that lacked NosI was also prepared and treated identically. NosK was separated from other proteins on a 12% SDS-PAGE gel, and the corresponding band was excised. An in-gel trypsin digestion was performed on the excised band with sequencing-grade lyophilized trypsin and subsequently analyzed at the Penn State Proteomics and Mass Spectrometry Core facility.

Mass spectra of tryptic digests were acquired using the default instrument parameters for the reflector positive-ion detection over the 700–3000 m/z range, and the method was calibrated with a bovine serum albumin tryptic digest standard (Protea Biosciences). Mass spectra were opened in the Bruker FlexAnalysis, smoothed by Savitzky–Golay filtering (0.2 m/z, 1 cycle), and baseline-subtracted using Top-Hat filtering. The mass lists were generated using a SNAP peak detection algorithm with a signal-to-noise threshold set at 6 and using the averagine SNAP average composition. The peak at m/z 1581.733 was selected for TOF-TOF analysis to establish the modification position. The resulting MS² data were processed using the default parameters and loaded into the Bruker Biotools application. The fragment ions were identified based on the best match between the experimental mass spectra and the peak assignments generated by the Sequence Editor within Biotools.

HPLC Analysis of NosK Acylation

The complete reaction contained the following in a total volume of 30 μL: 250 μM NosK, 50 μM NosJ, 25 μM NosI, 10 μM Ec holo ACP synthase, 2 mM ATP, 2 mM MIA, 100 μM CoA, and 25 mM HEPES, pH 7.5. For some reactions, various components of the complete reaction mixture were omitted as indicated in the text and legend for Figure ​Figure66. NosK control consisted of 250 μM NosK, 100 mM MgCl₂, and 25 mM HEPES, pH 7.5. The control acylated NosK reaction consisted of 250 μM NosK, 50 μM NosJ, 10 μM Ec Holo ACP synthase, 2 mM ATP, 2 mM MIA, 100 μM CoA, and 25 mM HEPES pH 7.5. The NosK S102A reaction contained 250 μM NosK S102A, 50 μM NosJ, 25 μM NosI, 10 μM Ec Holo ACP synthase, 2 mM ATP, 2 mM MIA, 100 μM CoA, and 25 mM HEPES pH 7.5. The reactions were in a total volume of 30 μL and were incubated at room temperature for approximately 1 h. Before analysis by HPLC, the reactions were quenched with 0.2% TFA pH 2.4.

Samples were analyzed using the Agilent 1100 series HPLC ChemStation software. A Zorbax 300 stable bond C18 5 μm 4.6 mm × 250 mm column was used with two different solvents: solvent A, 0.2% TFA pH 2.4 and solvent B, acetonitrile flowed at 1.4 mL per minute. The column was equilibrated in 45% solvent B and 55% solvent A, which was maintained for 3 min after injection. From 3 to 13 min, solvent B was increased to 57%. From 13 to 14 min, solvent B was increased to 100%. From 14 to 16 min, solvent B was held constant at 100%. From 16 to 17 min, solvent B was decreased to 45%. From 17 to 18 min, solvent B was held constant at 45%.

We thank Drs. Tatiana Laremore and James Miller of the Penn State Proteomics and Mass Spectrometry Core Facility, University Park, PA, for performing mass spectrometric analyses. We also thank Dr. Neela Yennawar of the Huck Institutes of Life Sciences or her help with X-ray structure determination. This research used resources of the Advanced Photon Source, a U.S. Department of Energy Office (DOE) of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. S.J.B. is an investigator of the Howard Hughes Medical Institute. This work was supported by NIH Grants GM-101957, GM 103268, and GM-122595, Penn State funds for undergraduate research, and a grant from the Pennsylvania Department of Health (TSR13/14 SAP 4100062216), all to S.J.B. It was also supported by an NIH K99/R00 Pathway to Independence award to A.K.B. (GM-100011), and a Rodney Erickson Discovery Grant for undergraduate research to E.D.B.