The Structure of l-Tyrosine 2,3-Aminomutase from the C-1027 Enediyne Antitumor Antibiotic Biosynthetic Pathway^,

Abstract

The SgcC4 l-tyrosine 2,3-aminomutase (SgTAM) catalyzes the formation of (S)-beta-tyrosine in the biosynthetic pathway of the enediyne antitumor antibiotic C-1027. SgTAM is homologous to the histidine ammonia lyase family of enzymes whose activity is dependent on the methylideneimidazole-5-one (MIO) cofactor. Unlike the lyase enzymes, SgTAM catalyzes additional chemical transformations resulting in an overall stereospecific 1,2-amino shift in the substrate l-tyrosine to generate (S)-beta-tyrosine. Previously, we provided kinetic, spectroscopic, and mutagenesis data supporting the presence of MIO in the active site of SgTAM [Christenson, S. D.; Wu, W.; Spies, A.; Shen, B.; and Toney, M. D. (2003) Biochemistry 42, 12708-12718]. Here we report the first X-ray crystal structure of an MIO-containing aminomutase, SgTAM, and confirm the structural homology of SgTAM to ammonia lyases. Comparison of the structure of SgTAM to the l-tyrosine ammonia lyase from Rhodobacter sphaeroides provides insight into the structural basis for aminomutase activity. The results show that SgTAM has a closed active site well suited to retain ammonia and minimize the formation of lyase elimination products. The amino acid determinants for substrate recognition and catalysis can be predicted from the structure, setting the framework for detailed mechanistic investigations.

Structural and Biochemical Characterization of the Therapeutic Anabaena variabilis Phenylalanine Ammonia Lyase

“…The recently published structure of tyrosine ammonia mutase also reported the existence of unexpected density which they assumed was from a reaction between the MIO group and β-mercaptoethanol that was used in sample preparation. 19 Regardless of whether the extra electron density in the active site contributes to stabilization of the active site, the presence of covalent modifications and stable excess electron density within this region highlight the reactive nature of the electrophile within the active site cavity.…”

A free-standing condensation enzyme catalyzing ester bond formation in C-1027 biosynthesis

“…1 A), which includes generation of (S)-␤-tyrosine by the aminomutase SgcC4 (23,24), activation and loading of (S)-␤-tyrosine to SgcC2 by the A enzyme SgcC1 (25,26), and halogenation and hydroxylation of (S)-␤-tyrosyl-SSgcC2 by the FAD-dependent SgcC3 halogenase and SgcC hydroxylase, respectively (27,28). We now report that SgcC5 does indeed catalyze ester bond formation between the SgcC2-tethered (S)-3-chloro-5-hydroxy-␤-tyrosine and (R)-1-phenyl-1,2-ethanediol, an enediyne core analog that mimics the aromatized C-1027 structure (structure 1a; Figs.…”

A new member of the 4-methylideneimidazole-5-one–containing aminomutase family from the enediyne kedarcidin biosynthetic pathway

“…Subsequent biochemical characterizations of SgcC4 have established that it (i) specifically catalyzes the conversion of L-tyrosine to (S)-β-tyrosine, shunting the primary metabolite into the C-1027 biosynthetic pathway, and (ii) releases 4-hydroxycinnamic acid as an intermediate, spuriously revealing weak activity as an ammonia lyase (7,8). Crystal structures have been solved for SgcC4 either alone or with mechanism-based inhibitors, as well as for the active site mutant SgcC4 (Tyr70Phe) with bound L-tyrosine (9)(10)(11)(12). These structures (i) unambiguously demonstrate the presence of the MIO prosthetic group, (ii) define the active site residues responsible for catalysis and substrate binding, and (iii) in comparison with structures of MIO-containing ammonia lyases, provide a method to bioinformatically differentiate MIO-containing aminomutases from ammonia lyases (3)(4)(5).…”

“…Mechanistic and structural characterizations of the MIO-containing aminomutases as a family have unveiled much unique chemistry, enzymology, and structural biology (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20), and exploitation of these enzymes as biocatalysts has provided access to α-and β-amino acids, which are difficult to prepare by other means (4,37). However, ʟ-phenylalanine and ʟ-tyrosine remain the only two known natural substrates (3)(4)(5).…”

“…Although KedY4 has shown no activity toward L-tyrosine and 2-aza-L-phenylalanine, the limited availability of other pyridine-containing L-α-amino acids has precluded testing of KedY4 substrate promiscuity; the similar kinetic parameters of KedY4 and SgcC4 would support such an exploration. The specificity of KedY4 and SgcC4 for 2-aza-Ltyrosine and L-tyrosine stems from a conserved set of polar residues lining the end of the substrate binding pocket that make key hydrogen bonds to the phenol group, as defined in the crystal structures of SgcC4 (9)(10)(11)(12). The corresponding residues in the PAMs are hydrophobic in nature (15,18).…”