Substrate Specificity of the Adenylation Enzyme SgcC1 Involved in the Biosynthesis of the Enediyne Antitumor Antibiotic C-1027
2006; Elsevier BV; Volume: 281; Issue: 40 Linguagem: Inglês
10.1074/jbc.m605887200
ISSN1083-351X
AutoresSteven G. Van Lanen, Shuangjun Lin, Pieter C. Dorrestein, Neil L. Kelleher, Ben Shen,
Tópico(s)Sesquiterpenes and Asteraceae Studies
ResumoC-1027 is an enediyne antitumor antibiotic composed of a chromophore with four distinct chemical moieties, including an (S)-3-chloro-4,5-dihydroxy-β-phenylalanine moiety that is derived from l-α-tyrosine. SgcC4, a novel aminomutase requiring no added co-factor that catalyzes the formation of the first intermediate (S)-β-tyrosine and subsequently SgcC1 homologous to adenylation domains of nonribosomal peptide synthetases, was identified as specific for the SgcC4 product and did not recognize any α-amino acids. To definitively establish the substrate for SgcC1, a full kinetic characterization of the enzyme was performed using amino acid-dependent ATP-[32P]PPi exchange assay to monitor amino acid activation and electrospray ionization-Fourier transform mass spectroscopy to follow the loading of the activated β-amino acid substrate to the peptidyl carrier protein SgcC2. The data establish (S)-β-tyrosine as the preferred substrate, although SgcC1 shows promiscuous activity toward aromatic β-amino acids such as β-phenylalanine, 3-chloro-β-tyrosine, and 3-hydroxy-β-tyrosine, but all were <50-fold efficient. A putative active site mutant P571A adjacent to the invariant aspartic acid residue of all α-amino acid-specific adenylation domains known to date was prepared as a preliminary attempt to probe the substrate specificity of SgcC1; however the mutation resulted in a loss of activity with all substrates except (S)-β-tyrosine, which was 142-fold less efficient relative to the wild-type enzyme. In total, SgcC1 is now confirmed to catalyze the second step in the biosynthesis of the (S)-3-chloro-4,5-dihydroxy-β-phenylalanine moiety of C-1027, presenting downstream enzymes with an (S)-β-tyrosyl-S-SgcC2 thioester substrate, and represents the first β-amino acid-specific adenylation enzyme characterized biochemically. C-1027 is an enediyne antitumor antibiotic composed of a chromophore with four distinct chemical moieties, including an (S)-3-chloro-4,5-dihydroxy-β-phenylalanine moiety that is derived from l-α-tyrosine. SgcC4, a novel aminomutase requiring no added co-factor that catalyzes the formation of the first intermediate (S)-β-tyrosine and subsequently SgcC1 homologous to adenylation domains of nonribosomal peptide synthetases, was identified as specific for the SgcC4 product and did not recognize any α-amino acids. To definitively establish the substrate for SgcC1, a full kinetic characterization of the enzyme was performed using amino acid-dependent ATP-[32P]PPi exchange assay to monitor amino acid activation and electrospray ionization-Fourier transform mass spectroscopy to follow the loading of the activated β-amino acid substrate to the peptidyl carrier protein SgcC2. The data establish (S)-β-tyrosine as the preferred substrate, although SgcC1 shows promiscuous activity toward aromatic β-amino acids such as β-phenylalanine, 3-chloro-β-tyrosine, and 3-hydroxy-β-tyrosine, but all were <50-fold efficient. A putative active site mutant P571A adjacent to the invariant aspartic acid residue of all α-amino acid-specific adenylation domains known to date was prepared as a preliminary attempt to probe the substrate specificity of SgcC1; however the mutation resulted in a loss of activity with all substrates except (S)-β-tyrosine, which was 142-fold less efficient relative to the wild-type enzyme. In total, SgcC1 is now confirmed to catalyze the second step in the biosynthesis of the (S)-3-chloro-4,5-dihydroxy-β-phenylalanine moiety of C-1027, presenting downstream enzymes with an (S)-β-tyrosyl-S-SgcC2 thioester substrate, and represents the first β-amino acid-specific adenylation enzyme characterized biochemically. C-1027 is an enediyne antitumor antibiotic isolated from the fermentation broth of Streptomyces globisporus (1Hu J. Xue Y.C. Xie M.Y. Zhang R. Otani T. Minami Y. Yamada Y. Marunaka T. J. Antibiot. (Tokyo). 1988; 41: 1575-1579Crossref PubMed Scopus (154) Google Scholar, 2Otani T. Minami Y. Marunaka T. Zhang R. Xie M.Y. J. Antibiot. (Tokyo). 1988; 41: 1580-1585Crossref PubMed Scopus (110) Google Scholar). It is produced as a chromoprotein complex consisting of a binding protein (annotated as CagA) and the reactive C-1027 chromophore containing a conjugated enediyne harbored within a nine-membered cyclic ring (see Fig. 1). Upon release from CagA, the enediyne core of the C-1027 chromophore readily undergoes a Bergman cycloaromatization to yield a transient biradical species capable of extracting hydrogen atoms from DNA, which in the presence of molecular oxygen can ultimately lead to single- and double-stranded DNA breaks (3Sugimoto Y. Otani T. Oie S. Wierzba K. Yamada Y. J. Antibiot. (Tokyo). 1990; 43: 417-421Crossref PubMed Scopus (89) Google Scholar). C-1027 and the entire enediyne family share this mode of action, and as a family, their potent cytotoxicity rivals that of any previously discovered natural product (4Wang X. Xie H. Drugs Future. 1999; 24: 847-852Crossref Scopus (12) Google Scholar). The biosynthetic gene cluster for C-1027 was previously cloned and sequenced, and analysis of the open reading frames (ORFs) 4The abbreviations used are: ORF, open reading frame; NRPS, nonribosomal peptide synthetase; PCP, peptidyl carrier protein; ESI-MS, electrospray ionization-mass spectrometry; ESI-FTMS, electrospray ionization-Fourier transform mass spectrometry; PheA, the excised l-Phe-specific adenylation domain of GrsA; ACES, 2-[(2-amino-2-oxoethyl)amino]ethanesulfonic acid. provided genetic evidence to adequately propose a mechanism of biosynthesis, transport, resistance, and regulation of C-1027 (5Liu W. Chrisenson S.D. Standage S. Shen B. Science. 2002; 297: 1170-1173Crossref PubMed Scopus (255) Google Scholar). Comparison of the gene cluster to other members of the enediyne family, including calicheamicin (6Ahlert J. Shepard E. Lomovskaya N. Zazopoulos E. Staffa A. Bachmann B.O. Huang K. Fonstein L. Czisny A. Whitwam R.E. Farnet C.M. Thorson J.S. Science. 2002; 297: 1173-1176Crossref PubMed Scopus (246) Google Scholar) and neocarzinostatin (7Liu W. Nonaka K. Nie L. Zhang J. Christenson S.D. Bae J. Van Lanen S.G. Zazopoulos E. Farnet C.M. Yang C.F. Shen B. Chem. Biol. 2005; 12: 1-10Abstract Full Text Full Text PDF PubMed Google Scholar), revealed a unified biosynthetic approach among the enediynes and supported a convergent biosynthesis of C-1027 from four components, a deoxy aminosugar, benzoxazolinate, β-amino acid, and enediyne core starting from glucose-1-phosphate, chorismic acid, l-α-tyrosine, and acyl-CoAs, respectively (Fig. 1). The pathway to generating the C-1027 deoxy aminosugar was initially targeted for biochemical analysis, and the first reaction catalyzed by SgcA1 has been characterized as a α-d-glucopyranosyl-1-phosphate thymidylyltransferase (8Murrell J.M. Liu W. Shen B. J. Nat. Prod. 2004; 67: 206-213Crossref PubMed Scopus (27) Google Scholar). Of the remaining moieties, only the initial step for the β-amino acid moiety (S)-3-chloro-4,5-dihydroxy-β-phenylalanine has been identified, a novel aminomutase reaction catalyzed by SgcC4 converting l-α-tyrosine to (S)-β-tyrosine (9Christenson S.D. Wu W. Spies M.A. Shen B. Toney M.D. Biochemistry. 2003; 42: 12708-12718Crossref PubMed Scopus (69) Google Scholar). Among the 56 ORFs identified within the boundaries of the C-1027 biosynthetic gene cluster, three ORFs exhibit high homology to nonribosomal peptide synthetase (NRPS) domains. NRPSs are typically large modular proteins that catalyze the synthesis of a wide range of biologically active peptides (10Schwarzer D. Finking R. Marahiel M.A. Nat. Prod. Rep. 2003; 20: 275-287Crossref PubMed Scopus (445) Google Scholar). A minimal NRPS module consists of three domains, an adenylation domain that sequentially selects, activates, and loads an amino acid (or carboxylic acid) to the 4′-phosphopantetheine prosthetic group of a peptidyl carrier protein (PCP) domain and a condensation domain that catalyzes the formation of an amide bond. In most examples, a single NRPS protein contains multiple modules, and the primary sequence of the resulting peptide product is correlated to the linear architecture of the modules (hence the so-called “thiotemplate co-linearity” rule). Extra domains embedded in the multimodular NRPS proteins, such as epimerization, methyltransferase, or oxidation domains, can be present to afford final peptides with enriched chemistry. As opposed to large multimodular proteins, singular domains or didomains of NRPS as individual ORFs have also been recently characterized, and this phenomenon has facilitated research toward understanding domain-domain recognition and interaction in NRPS and the molecular mechanism of NRPS-catalyzed nonribosomal peptide biosynthesis. Within the C-1027 biosynthetic gene cluster, two ORFs upstream and in opposite orientation to sgcC4 were found to encode proteins with sequence similarities to condensation (SgcC5) and PCP (SgcC2) domains (Fig. 2A). An ORF located adjacent to and downstream of sgcC4 was also identified and encodes an 881-amino-acid protein, SgcC1, having sequence homology to adenylation domains. Although SgcC1 has closest homology to adenylation domains that activate l-α-tyrosine, preliminary characterization has revealed that SgcC1 recognizes (S)-β-tyrosine as opposed to any of the standard α-amino acids, including either isomer of α-tyrosine or α-phenylalanine or analogs (11Van Lanen S.G. Dorrestein P.C. Christenson S.D. Liu W. Ju J. Kelleher N.L. Shen B. J. Am. Chem. Soc. 2005; 127: 11594-11595Crossref PubMed Scopus (49) Google Scholar). Although these results support the findings that SgcC4 is the initial enzyme in the pathway (catalyzing the stereospecific formation of (S)-β-tyrosine from l-α-tyrosine) and SgcC1 catalyzes the subsequent step (Fig. 2B), they contradicted the prediction on the basis of the so-called “nonribosomal codes” (12Stachelhaus T. Mootz H.D. Marahiel M.A. Chem. Biol. 1999; 6: 493-505Abstract Full Text PDF PubMed Scopus (974) Google Scholar, 13Challis G.L. Ravel J. Townsend C.A. Chem. Biol. 2000; 7: 211-224Abstract Full Text Full Text PDF PubMed Scopus (654) Google Scholar) that SgcC1 would be an α-amino acid-specific adenylation domain (11Van Lanen S.G. Dorrestein P.C. Christenson S.D. Liu W. Ju J. Kelleher N.L. Shen B. J. Am. Chem. Soc. 2005; 127: 11594-11595Crossref PubMed Scopus (49) Google Scholar). As a follow-up to the initial characterization of SgcC1, we now provide definitive experimental data to assign the substrate specificity for SgcC1. Using amino acid-dependent ATP-[32P]PPi exchange assays and electrospray ionization-Fourier transform mass spectrometry (ESI-FTMS), chlorinated and hydroxylated tyrosine or phenylalanine analogs were tested revealing that (S)-β-tyrosine is the preferred substrate for SgcC1. The results unambiguously establish that SgcC1, following the SgcC4 aminomutase reaction, catalyzes the second step in the biosynthesis of the (S)-3-chloro-4,5-dihydroxy-β-phenylalanine moiety from l-α-tyrosine by forming the (S)-β-tyrosyl-S-SgcC2 intermediate. Subsequent steps involve halogenation (SgcC3), hydroxylation (SgcC), and incorporation of the fully modified β-amino acid unit into the enediyne core (SgcC5), although the precise timing of the last steps awaits further validation (Fig. 2B). SgcC1 as a naturally occurring, discrete protein could therefore provide a relatively simple platform to address the inherent amino acid specificity-conferring elements of adenylation domains to β-amino acids, of which the current nonribosomal codes were based solely on a single structure of the excised, l-α-phenylalanine-specific adenylation domain of GrsA (PheA) (12Stachelhaus T. Mootz H.D. Marahiel M.A. Chem. Biol. 1999; 6: 493-505Abstract Full Text PDF PubMed Scopus (974) Google Scholar, 13Challis G.L. Ravel J. Townsend C.A. Chem. Biol. 2000; 7: 211-224Abstract Full Text Full Text PDF PubMed Scopus (654) Google Scholar). As an initial attempt to determine the structural elements of SgcC1 necessary for proper β-amino acid selection, truncated protein and a single point mutation of SgcC1 were generated and the results reported. Chemicals and Instrumentation—If not mentioned, chemicals and instruments used were identical to that previously reported (11Van Lanen S.G. Dorrestein P.C. Christenson S.D. Liu W. Ju J. Kelleher N.L. Shen B. J. Am. Chem. Soc. 2005; 127: 11594-11595Crossref PubMed Scopus (49) Google Scholar). Cinnamic acid, p-hydroxycinnamic acid, and 3-amino-3-phenylpropionic acid (β-phenylalanine) were from Fisher-Acros (Pittsburgh, PA). (R)-3-Amino-(4-hydroxy-phenyl)-propionic acid ((R)-β-tyrosine) was from PepTech Corporation (Burlington, MA). Restriction enzymes were from New England Biolabs (Beverly, MA) or Invitrogen, and expression vectors were from Novagen (Madison, WI). Unless explicitly stated, the compounds used were racemic mixtures. Protein analysis was performed with position-specific iterated-BLAST and BL2SEQ using the San Diego Supercomputer Center Biology WorkBench, version 3.2, and the adenylation domain active sites were extracted using the NRPS Predictor software from Universität Tübingen. Synthesis of Racemic 3-Chloro-β-tyrosine—Synthesis of 3-chloro-β-tyrosine was achieved by following the method reported by Weaver and co-worker (14Tan C.Y.K. Weaver D.F. Tetrahedron. 2002; 58: 7449-7461Crossref Scopus (63) Google Scholar). 3-Chloro-benzaldehyde (76 mg) was refluxed with 1 equivalent of malonic acid (51 mg) and 2 equivalents of ammonium acetate (76 mg) in ethanol (5 ml) for 2 days. The reaction mixture was adjusted to an approximate pH of 4 with 1 n HCl and separated by a strong acid cation exchange column (Dowex® 50WX8). The product, 3-chloro-β-tyrosine, was eluted with 1% ammonium hydroxide. The residue was dissolved in distilled water after the solvent was removed through evaporation under reduced pressure and further purified on C-18 reverse phase column (Alltima 10 × 250 mm, 5μm; Grace Davison Discovery Sciences). 1H NMR assignments in D2O containing CF3CO2D at 400 MHz are as follows (where d means doublet, dd means doublet of doublets, m means multiplet, and t means triplet): δ 7.08 (d, J = 2.4 Hz, 1H), 6.87 (dd, J = 8.8, 2.4 Hz, 1H), 6.65 (d, J = 8.4 Hz, 1H), 4.30 (t, J = 7.2 Hz, Hβ, 1H), 2.77 (dd, J = 17.2, 7.6 Hz, Hα, 1H), and 2.66 (dd, J = 17.2, 6.4 Hz, Hα, 1H). 13C NMR assignments in D2O at 100 MHz are as follows: δ 173.1 (C=O), 152.8 (ArC), 129.2 (ArC), 128.0 (ArC), 127.1 (ArC), 120.7 (ArC), 117.6 (ArC), 50.7 (Cβ), and 37.5 (Cα). ESI-MS gave m/z:[M + H]+, 216.0; [M – H]–, 213.9; calculated 215.0 for C9H10NO3Cl. Synthesis of Racemic 3-Hydroxy-β-tyrosine—3-Hydroxy-β-tyrosine was obtained by the same procedure as that used for the synthesis of 3-chloro-β-tyrosine beginning from 3-hydroxy-benzaldehyde. 1H NMR assignments in D2O at 400 MHz are as follows: δ 6.74–6.85 (m, 3H), 4.42 (t, J = 6.8 Hz, Hβ, 1H), 2.79 (dd, J = 1 5.6, 8.0 Hz, Hα, 1H), and 2.69 (dd, J = 16.0, 6.4 Hz, Hα, 1H). 13C NMR assignments in D2O at 125 MHz are as follows: δ 40.9 (Cα), 52.7 (Cβ), 115.0 (ArC), 116.6 (ArC), 119.4 (ArC), 128.7 (ArC), 145.6 (ArC), 145.5 (ArC), and 177.9 (C=O). ESI-MS gave m/z:[M + H]+, 198.0; [M – H]–, 195.9; calculated 197.0 for C9H11NO4. Synthesis of Racemic 3-Chloro-5-hydroxy-β-tyrosine—3-Chloro-5-hydroxy-β-tyrosine was synthesized by the same procedure as that used for the synthesis of 3-chloro-β-tyrosine beginning from 3-cloro-4,5-dihydroxybenzaldehyde, which was prepared from 3-chloro-4-hydroxyl-5-methoxybenzaldehyde following the method reported by Perchellet and co-workers (15Hua D.H. Huang X. Chen Y. Battina S.K. Tamura M.S. Noh K. Koo S.I. Namatame I. Tomoda H. Perchellet E.M. Perchellet J.P. J. Org. Chem. 2004; 69: 6065-6078Crossref PubMed Scopus (53) Google Scholar). 1H NMR assignments in D2O at 500 MHz are as follows: δ 7.04 (d, J = 2.5 Hz, 1H), 6.92 (d, J = 2.5 Hz, 1H), 4.60 (t, J = 7.5 Hz, 1H), 3.04 (dd, J = 17.0, 8.0 Hz, 1H), and 2.95 (dd, J = 17.0, 6.5 Hz, 1H). 13C NMR assignments in D2O at 125 MHz are as follows: δ 38.8 (Cα), 51.6 (Cβ), 113.6, 120.2, 121.8, 128.6, 141.8, 146.1 (ArC), and 175.1 (C=O). ESI-MS gave m/z: [M + H]+, 232.1; calculated 231.0 for C9H1oNO4Cl. DNA Manipulations—Cloning and construction of the sgcC1 (pBS1033) and sgcC2 (pBS1034) expression vectors were previously described (11Van Lanen S.G. Dorrestein P.C. Christenson S.D. Liu W. Ju J. Kelleher N.L. Shen B. J. Am. Chem. Soc. 2005; 127: 11594-11595Crossref PubMed Scopus (49) Google Scholar). A truncated form of SgcC1 was prepared by PCR amplification using pBS1005 (5Liu W. Chrisenson S.D. Standage S. Shen B. Science. 2002; 297: 1170-1173Crossref PubMed Scopus (255) Google Scholar) as a template and a forward primer of 5′-GGGAATTCCATATGGGCGCTCTGCCGCTGGAC-3′ (NdeI site underlined) and a reverse primer of 5′-GGCAAGCTTGCGGGTGAGCCGGGAGCG-3′ (HindIII site underlined). The amplified 1629-base-pair fragment was cloned into the same sites of pET29a to yield pBS1037. After confirmation of the DNA sequence fidelity, the NdeI/HindIII fragment was isolated and cloned into pET28a to yield pBS1038. Although the former construct produces SgcC1 as a C-terminal His6-tagged protein, the latter construct produces SgcC1 as a C- and N-terminal His6-tagged protein, both of which have the 338 amino acids at the N terminus of SgcC1 deleted. A P571A point mutation of SgcC1 was generated by PCR amplification of the template pBS1033 (11Van Lanen S.G. Dorrestein P.C. Christenson S.D. Liu W. Ju J. Kelleher N.L. Shen B. J. Am. Chem. Soc. 2005; 127: 11594-11595Crossref PubMed Scopus (49) Google Scholar) using the Expand long template PCR system (Roche Applied Science). Reactions were performed using the manufacturer's provided Buffer 2 with 5% Me2SO, primers of 5′-GTCTCCCCGGAGCACGACGCGGCGCTGGCCGAGGTC-3′ and the reverse complement (with the Ala codon underlined), and a PCR program consisting of an initial hold at 94 °C for 2 min followed by 20 cycles of 94 °C for 10 s, 56 °C for 30 s, and 68 °C for 7 min. The template DNA was digested with 10 units of DpnI for 1 h at 37 °C followed by heating to 90 °C for 5 min and cooling at room temperature before transformation. The introduction of the correct point mutation and the fidelity of the entire gene including 250 bp upstream and downstream were confirmed by DNA sequencing to yield pBS1039. Amino Acid-dependent ATP-[32P]PPi Exchange Assays—Assessment of the SgcC1 adenylation enzyme substrate specificity was performed as described previously (11Van Lanen S.G. Dorrestein P.C. Christenson S.D. Liu W. Ju J. Kelleher N.L. Shen B. J. Am. Chem. Soc. 2005; 127: 11594-11595Crossref PubMed Scopus (49) Google Scholar). A pH profile for SgcC1 was generated using a three-buffer system of 52/52/100 mm Tris-ACES-ethanolamine as described previously (16Ellis K.J. Morrison J.F. Methods Enzymol. 1982; 87: 405-426Crossref PubMed Scopus (652) Google Scholar). Assays were run at 30 °C in 1× buffer with 5 mm MgCl2, 0.1 mm EDTA, 5 mm ATP, 0.9 μm [32P]PPi, 1 mm (S)-β-tyrosine, and 10 nm SgcC1 under initial velocity conditions (<10% isotopic conversion into ATP). Each data point represents a minimum of four duplicate end-point assays. Because the pH profile was utilized only for determining the conditions for optimal activity, the data were fitted to a smooth curve in contrast to a specific rate equation, which would warrant further kinetic analysis at varied pH. The steady-state kinetic parameters for SgcC1 were determined with activity assays carried out at 30 °C in 100 mm Tris-HCl (pH 9.0), 5 mm MgCl2, 0.1 mm EDTA, 5 mm ATP, 1.0 μm [32P]PPi, and varied co-substrate as follows: 25–1600 μm for 3-chloro-β-tyrosine, 3-hydroxy-β-tyrosine, and β-phenylalanine, 5–370 μm for (R)-β-tyrosine, and 0.5–200 μm for (S)-β-tyrosine. Enzyme concentration for each substrate ranged from 25 to 70 nm to maintain initial velocity conditions. Single time points were analyzed between 1 and 5 min; each data point represents a minimum of four duplicates with a S.D. of 50-fold more efficient with (S)-β-tyrosine than any substrate analog tested, excluding the R-enantiomer, which had a 25-fold lower efficiency.TABLE 1Michaelis-Menten constants for SgcC1 as an adenylation enzyme toward selected β-amino acidsSubstrateKmkcatkcat/KmRelative efficiencyμms-1μm-1s-1(S)-β-Tyr3.2 ± 0.62.2 ± 0.50.691(R)-β-Tyr53 ± 121.5 ± 0.30.0280.043-OH-β-Tyr81 ± 161.2 ± 0.10.0150.02β-Phe1.9 × 102 ± 401.4 ± 0.40.00750.013-Cl-β-Tyr3.0 × 102 ± 501.6 ± 0.30.00530.008 Open table in a new tab SgcC1-catalyzed Loading of SgcC2—The second half-reaction of SgcC1 was monitored by ESI-FTMS following the mass shift observed upon loading the activated β-tyrosine onto holo-SgcC2 PCP to generate a β-tyrosyl-S-SgcC2 thioester. With either (S)- or (R)-β-tyrosine as the substrate, the appropriate mass shift of +162.6 was observed (11Van Lanen S.G. Dorrestein P.C. Christenson S.D. Liu W. Ju J. Kelleher N.L. Shen B. J. Am. Chem. Soc. 2005; 127: 11594-11595Crossref PubMed Scopus (49) Google Scholar). Time course analysis with (S)-β-tyrosine resulted in a rate constant of 2.1 s–1 and with a (R)-β-tyrosine rate constant of 1.9 s–1, both of which are nearly identical to the kcat calculated from the first half-reaction as determined by the ATP-[32P]PPi exchange assays (Table 1). SgcC1 Mutation Analysis—Adenylation domains have been proposed to extend ∼550 amino acids within an NRPS module (10Schwarzer D. Finking R. Marahiel M.A. Nat. Prod. Rep. 2003; 20: 275-287Crossref PubMed Scopus (445) Google Scholar). Therefore, the wild-type SgcC1 protein (881-amino-acid residues) was truncated by deleting 338 amino acids from its N terminus to encompass only the putative AMP-forming domain (C terminus). Of the constructs prepared, only the dual His6-tagged version was slightly soluble, but no activity was observed with this construct. A P571A mutant of SgcC1 was prepared to examine possible changes in substrate specificity and/or catalysis. This residue neighbors the invariant aspartate residue Asp-570, the carboxylate side chain of which interacts with the α-amino group to lock the orientation of the l-α-amino acid into the substrate binding pocket (Fig. 3). The mutation abolished activity with all substrates except (S)-β-tyrosine. Kinetic analysis for SgcC1(P571A) yielded a Km of 13 ± 4 μm and a kcat of 0.07 ± 0.02 s–1, a 142-fold loss in catalytic efficiency when compared with the wild-type SgcC1 (Table 2).TABLE 2Michaelis-Menten constants for SgcC1(P571A) as an adenylation enzyme toward (S)-β-tyrosine and comparison with that of the wild-type SgcC1 enzymeEnzymeKmkcatkcat/KmRelative efficiencyμms-1μm-1s-1SgcCl (wild-type)3.2 ± 0.62.2 ± 0.50.691SgcCl (P576A)13 ± 47.0 × 10-2 ± 0.020.00540.007 Open table in a new tab Adenylation domains of NRPS catalyze the activation, at the expense of ATP, and subsequent loading of the activated amino acids or carboxylic acids to a PCP domain to form an aminoacyl-S-PCP thioester. SgcC1 has significant sequence homology to numerous adenylation domains of NRPS, and as a result, has been hypothesized to be involved in the formation, activation, and incorporation of the (S)-3-chloro-4,5-dihydroxy-β-phenylalanine moiety into C-1027, although the precise timing of the steps cannot be predicted a priori (Fig. 2). Sequence analysis predicted l-α-tyrosine as the probable substrate for SgcC1, as deduced from its closest sequence identity to domains that activate l-α-tyrosine, such as TycC3, NovH, and SimH, and comparisons to the nonribosomal codes of NRPS suggested that l-α-tyrosine was the most likely substrate for SgcC1 (Fig. 3) (12Stachelhaus T. Mootz H.D. Marahiel M.A. Chem. Biol. 1999; 6: 493-505Abstract Full Text PDF PubMed Scopus (974) Google Scholar, 13Challis G.L. Ravel J. Townsend C.A. Chem. Biol. 2000; 7: 211-224Abstract Full Text Full Text PDF PubMed Scopus (654) Google Scholar). However, our previous results with SgcC4 preferring l-α-tyrosine were inconsistent with this proposal (11Van Lanen S.G. Dorrestein P.C. Christenson S.D. Liu W. Ju J. Kelleher N.L. Shen B. J. Am. Chem. Soc. 2005; 127: 11594-11595Crossref PubMed Scopus (49) Google Scholar), and therefore, a full biochemical characterization of SgcC1 was undertaken. As previously reported, when l-α-tyrosine was tested as a substrate, SgcC1 was only active in the presence of SgcC4, which converts l-α-tyrosine t
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