Biocatalytic Carbon–Hydrogen and Carbon–Fluorine Bond Cleavage through Hydroxylation Promoted by a Histidyl-Ligated Heme Enzyme

Abstract

LmbB2 is a peroxygenase-like enzyme that hydroxylates L-tyrosine to L-3,4-dihydroxyphenylalanine (DOPA) in the presence of hydrogen peroxide. However, its heme cofactor is ligated by a proximal histidine, not cysteine. We show that LmbB2 can oxidize L-tyrosine analogs with ring-deactivated substituents such as 3-nitro-, fluoro-, chloro-, iodo-L-tyrosine. We also found that the 4-hydroxyl group of the substrate is essential for reacting with the heme-based oxidant and activating the aromatic C-H bond. The most interesting observation of this study was obtained with 3-fluoro-L-tyrosine as a substrate and mechanistic probe. The LmbB2-mediated catalytic reaction yielded two hydroxylated products with comparable populations, i.e., oxidative C-H bond cleavage at C5 to generate 3-fluoro-5-hydroxyl-L-tyrosine and oxygenation at C3 concomitant with a carbon-fluorine bond cleavage to yield DOPA and fluoride. An iron protein-mediated hydroxylation on both C-H and C-F bonds with multiple turnovers is unprecedented. Thus, this finding reveals a significant potential of biocatalysis in C-H/C-X bond (X = halogen) cleavage. Further 18O-labeling results suggest that the source of oxygen for hydroxylation is a peroxide, and that a commonly expected oxidation by a high-valent iron intermediate followed by hydrolysis is not supported for the C-F bond cleavage. Instead, the C-F bond cleavage is proposed to be initiated by a nucleophilic aromatic substitution mediated by the iron-hydroperoxo species. Based on the experimental results, two mechanisms are proposed to explain how LmbB2 hydroxylates the substrate and cleaves C-H/C-F bond. This study broadens the understanding of heme enzyme catalysis and sheds light on enzymatic applications in medicinal and environmental fields.

A novel catalytic heme cofactor in SfmD with a single thioether bond and a bis-His ligand set revealed by a de novo crystal structural and spectroscopic study

“…, the b -type heme-containing tyrosine hydroxylase, LmbB2. 10 Collectively, these results indicated that the loss of the single cysteine–vinyl covalent linkage through mutation led to heme with an electronic structure reminiscent of b -type heme and results in a catalytically inactive variant.…”

“…7 SfmD requires a mononuclear heme as its prosthetic group for oxygenation, similar to enzymes with analogous activity, i.e., heme-based tyrosine hydroxylases (TyrH), 8 including LmbB2 and Orf13. [9][10][11] Such a cofactor dependency for aromatic amino acid oxidation is mechanistically distinct from other types of tyrosine hydroxylation mediated by nonheme-dependent monooxygenases such as pterin-dependent tyrosine hydroxylase, 12,13 copper-dependent tyrosinase, 14 and the two-component, FADdependent monooxygenase found in SgcC. 15 SfmD was previously reported as a heme peroxidase with an atypical peroxidase activity of hydroxylating 3-Me-L-Tyr or L-Tyr using hydrogen peroxide as oxidant.…”

Enzymatic defluorination of fluorinated compounds

“…At the same time 3-fluoro-5-hydroxyl-l-tyrosine is also produced by oxidative C-H bond cleavage at C5 in the same reaction mediated by LmbB2 (Fig. 8) [37].…”

“…Peroxygenase-like LmbB2 (histidyl-ligated heme enzyme) coded by the lmbB2 gene of the lincomycin biosynthesis gene cluster in Streptomyces lincolnensis catalyzes the defluorination of 3-fluorotyrosine to yield 3,4-dihydroxyphenylalanine (DOPA) under the presence of H 2 O 2 (Fig. 8) [37]. At the same time 3-fluoro-5-hydroxyl-l-tyrosine is also produced by oxidative C-H bond cleavage at C5 in the same reaction mediated by LmbB2 (Fig.…”

Enzyme-catalyzed C–F bond formation and cleavage

“…Although the C-F bond is the strongest covalent single bond in organic chemistry, cysteine dioxygenase is able to cleave a C-F bond to realize co-factor biogenesis (see Scheme 5a). In 2019, Wang's group discovered that a histidine-linked heme enzyme can catalyze the cleavage of C-F by hydroxylation (Wang et al 2019). LmbB2 has a wide range of catalytic activities toward l-tyrosine analogues, as long as the 4-hydroxyl group is present.…”