Design and characterization of mechanism-based inhibitors for the tyrosine aminomutase SgTAM

Montavon, Timothy J.; Christianson, Carl V.; Festin, Grace M; Shen, Ben; Bruner, Steven D.

doi:10.1016/j.bmcl.2007.11.046

Cited by 31 publications

(45 citation statements)

References 19 publications

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“…Such a structure could be essential for flexibility and plasticity of the enzyme and seems to be a functional requirement for substrate binding and catalysis. Data indicated that TbPAM has the same overall protein fold as tyrosine aminomutase (Sg-TAM) that was reported by Montavon and coworkers (11). Moreover, superposition of the theoretical structure of TbPAM with TAM and PAL indicated significant homology where TbPAM contains a 4-methylidene-imidazole-5-one (MIO) cofactor.…”

Section: Predicted Structure and Active Site Of Tbpamsupporting

confidence: 63%

Molecular Cloning and Characterization of the Phenylalanine Aminomutase Gene From Taxus baccata L.

2013

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Background: Taxol is one of the most important anti-cancer drugs, which is obtained from yew trees (Taxus sp.). T+he first step in side chain assembly of taxol is catalyzed by phenylalanine aminomutase, which converts α-phenylalanine to β-phenylalanine. Objectives: In this study, for the first time, we report on the cloning, preliminary expression and characterization of a full-length gene and cDNA encoding phenylalanine aminomutase from Taxus baccata L. Materials and Methods:Comparison of the full-length gene with other ones identified from the Taxus species showed high similarity, particularly with Taxus x media. Results:The results showed that the expression level of this gene in Taxus baccata is very low and therefore this enzymatic step could be a rate limiting step in the taxol biosynthesis pathway. Successful amplification of the cDNA was only obtained from RNA samples isolated from methyl jasmonate elicited suspension cells of Taxus baccata. The cloned cDNA contained a 2064 bp open reading frame encoding a protein composed of 687 amino acids. Sequence comparison analysis revealed that the gene is very similar (98 -99 %) with respect to the nucleotide and amino acid sequences in different Taxus species and also share the signature active site motif (175ASG177). Conclusions: The predicted structure of TbPAM was analyzed using bioinformatic tools. The results indicated that the protein has similar overall folding to tyrosine aminomutase.

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Section: Predicted Structure and Active Site Of Tbpamsupporting

confidence: 63%

Molecular Cloning and Characterization of the Phenylalanine Aminomutase Gene From Taxus baccata L.

2013

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“…Strong evidence for this latter mechanism comes from X-ray crystallographic work on TAM that had been inactivated by tyrosine analogues in which the a-hydrogens are substituted with fluorine atoms. [20,27] The use of these compounds, which cannot undergo ammonia elimination resulted in the formation of covalent adducts between MIO and the amino group of the analogues. This suggests that MIO might be coupled with the amino group of the substrate in the initial stage of the aminomutase reaction.…”

Section: Discussionmentioning

confidence: 99%

Enzymatic Synthesis of Enantiopure α‐ and β‐Amino Acids by Phenylalanine Aminomutase‐Catalysed Amination of Cinnamic Acid Derivatives

Szymański

Wietzes

et al. 2009

ChemBioChem

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The phenylalanine aminomutase (PAM) from Taxus chinensis catalyses the conversion of alpha-phenylalanine to beta-phenylalanine, an important step in the biosynthesis of the N-benzoyl phenylisoserinoyl side-chain of the anticancer drug taxol. Mechanistic studies on PAM have suggested that (E)-cinnamic acid is an intermediate in the mutase reaction and that it can be released from the enzyme's active site. Here we describe a novel synthetic strategy that is based on the finding that ring-substituted (E)-cinnamic acids can serve as a substrate in PAM-catalysed ammonia addition reactions for the biocatalytic production of several important beta-amino acids. The enzyme has a broad substrate range and a high enantioselectivity with cinnamic acid derivatives; this allows the synthesis of several non-natural aromatic alpha- and beta-amino acids in excellent enantiomeric excess (ee >99 %). The internal 5-methylene-3,5-dihydroimidazol-4-one (MIO) cofactor is essential for the PAM-catalysed amination reactions. The regioselectivity of amination reactions was influenced by the nature of the ring substituent.

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“…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).…”

Section: Streptomyces Globisporusmentioning

confidence: 99%

“…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).…”

Section: ·Smentioning

confidence: 99%

“…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).…”

Section: ·Smentioning

confidence: 99%

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

Huang¹,

Lohman²,

Huang³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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4-Methylideneimidazole-5-one (MIO)-containing aminomutases catalyze the conversion of l -α-amino acids to β-amino acids with either an ( R ) or an ( S ) configuration. l -Phenylalanine and l -tyrosine are the only two natural substrates identified to date. The enediyne chromophore of the chromoprotein antitumor antibiotic kedarcidin (KED) harbors an ( R )-2-aza-3-chloro-β-tyrosine moiety reminiscent of the ( S )-3-chloro-5-hydroxy-β-tyrosine moiety of the C-1027 enediyne chromophore, the biosynthesis of which uncovered the first known MIO-containing aminomutase, SgcC4. Comparative analysis of the KED and C-1027 biosynthetic gene clusters inspired the proposal for ( R )-2-aza-3-chloro-β-tyrosine biosynthesis starting from 2-aza- l -tyrosine, featuring KedY4 as a putative MIO-containing aminomutase. Here we report the biochemical characterization of KedY4, confirming its proposed role in KED biosynthesis. KedY4 is an MIO-containing aminomutase that stereospecifically catalyzes the conversion of 2-aza- l -tyrosine to ( R )-2-aza-β-tyrosine, exhibiting no detectable activity toward 2-aza- l -phenylalanine or l -tyrosine as an alternative substrate. In contrast, SgcC4, which stereospecifically catalyzes the conversion of l -tyrosine to ( S )-β-tyrosine in C-1027 biosynthesis, exhibits minimal activity with 2-aza- l -tyrosine as an alternative substrate but generating ( S )-2-aza-β-tyrosine, a product with the opposite stereochemistry of KedY4. This report of KedY4 broadens the scope of known substrates for the MIO-containing aminomutase family, and comparative studies of KedY4 and SgcC4 provide an outstanding opportunity to examine how MIO-containing aminomutases control substrate specificity and product enantioselectivity.

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Design and characterization of mechanism-based inhibitors for the tyrosine aminomutase SgTAM

Cited by 31 publications

References 19 publications

Molecular Cloning and Characterization of the Phenylalanine Aminomutase Gene From Taxus baccata L.

Molecular Cloning and Characterization of the Phenylalanine Aminomutase Gene From Taxus baccata L.

Enzymatic Synthesis of Enantiopure α‐ and β‐Amino Acids by Phenylalanine Aminomutase‐Catalysed Amination of Cinnamic Acid Derivatives

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

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