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

Abstract

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.

Keywords: Streptoalloteichus sp.; Streptomyces globisporus; amonia lyase; enzyme; natural product.

Fig. 1.

Comparison of biosynthetic pathways for the β-amino acid moieties of the C-1027 and KED chromophores featuring the MIO-containing aminomutases SgcC4 and KedY4. Shown are proposed biosynthetic pathways for the (S)-3-chloro-5-hydroxy-β-tyrosine moiety of C-1027 from ʟ-tyrosine (A) and the (R)-2-aza-3-chloro-β-tyrosine moiety of KED from 2-aza-ʟ-tyrosine (B).

Fig. 2.

Characterization of KedY4 as an MIO-containing aminomutase. (A) KedY4 catalyzed formation of (R)-2-aza-β-tyrosine and 2-aza-4-hydroxycinnamic acid from 2-aza-ʟ-tyrosine in comparison with SgcC4. (B) HPLC analysis of KedY4- and SgcC4-catalzyed forward reactions: (I) boiled KedY4 with 2-aza-ʟ-tyrosine, (II) KedY4 with 2-aza-ʟ-tyrosine, (III) SgcC4 with 2-aza-ʟ-tyrosine, (IV) KedY4 with ʟ-tyrosine, and (V) KedY4 with 2-aza-ʟ-phenylalanine. (C) HPLC analysis of KedY4- and SgcC4-catalzyed reverse reactions: (I) boiled KedY4 with (R)-2-aza-β-tyrosine, (II) KedY4 with (R)-2-aza-β-tyrosine, (III) KedY4 with (S)-2-aza-β-tyrosine, (IV) SgcC4 with (S)-2-aza-β-tyrosine, and (V) SgcC4 with (R)-2-aza-β-tyrosine. ●, 2-aza-ʟ-tyrosine; ▽, ʟ-tyrosine; ▼, 2-aza-ʟ-phenylalanine; ◆, (R)-2-aza-β-tyrosine; ◇, (S)-2-aza-β-tyrosine; ○, 2-aza-4-hydroxycinnamic acid. Note that the peaks for 2-aza-4-hydroxycinnamic acid reflect a minor product, because its extinction coefficient is much larger than those of 2-aza-ʟ-tyrosine and 2-aza-β-tyrosine at 280 nm.

Fig. 3.

Determination of KedY4-catalyzed formation of (R)-2-aza-β-tyrosine from 2-aza-ʟ-tyrosine proceeding with high enantiospecificity. (A) Synthesis of (S)- and (R)-MTPAs from KedY4-generated (R)-2-aza-β-tyrosine methyl ester. (B) Synthesis of (S)- and (R)-MTPAs from synthetic (S)-2-aza-β-tyrosine methyl ester. (C) ¹H NMR chemical shift differences Δδ^SR (in ppm) for (S)- and (R)-MTPAs of (R)- and (S)-2-aza-β-tyrosine methyl esters establishing the absolute configuration at C-3′. (D) Expanded ¹H NMR spectrum of (S)-MTPA of the KedY4-generated (R)-2-aza-β-tyrosine methyl ester showing H-3 at δ_H 8.40, with the doublet at δ_H 8.37, indicating the presence of (S)-MTPA of (S)-2-aza-β-tyrosine methyl ester as a minor product.

Fig. 4.

Kinetic analysis of KedY4 with 2-aza-ʟ-tyrosine as a substrate for the forward reaction and (R)-2-aza-β-tyrosine as a substrate for the reverse reaction. (A and B) Formation of (R)-2-aza-β-tyrosine (◆; A) and 2-aza-4-hydroxycinnamic acid (○; B) with varying amounts of 2-aza-ʟ-tyrosine. (C and D) Formation of 2-aza-ʟ-tyrosine (●; C) and 2-aza-4-hydroxycinnamic acid (D) with varying amounts of (R)-2-aza-β-tyrosine.