Homoprotocatechuate 2,3-Dioxygenase from Brevibacterium fuscum

A two-electron-shell game: intermediates of the extradiol-cleaving catechol dioxygenases

“…They catalyze the cleavage of the C2-C3 bond (Scheme 1A; See Scheme 2 for numbering convention) of homoprotocatechuate (HPCA) with the incorporation of both oxygen atoms of O 2 into the ring-cleaved product [13]. Crystal structures of the iron-containing enzyme from Brevibacterium fuscum (HPCD) and the manganese-containing enzyme from Arthrobacter globiformis (MndD) have been solved and reveal a remarkable structural similarity [8, 14, 15].…”

“…Steady-state kinetic measurements show that the two enzymes have similar k cat , K M O2 , and K M HPCA values for HPCA turnover as shown in Table 1 [8, 13, 23]. The likelihood that the metal center in both enzymes can transfer electron density to O 2 as it binds to the metal during the activation process presents an intriguing mechanistic conundrum.…”

“…The application of intermediate trapping and structural characterization in crystallo is already showing that very similar species are found for HPCD [13] and homogentisate dioxygenase [47]. To date, transient kinetic studies of only a few active site variants of HPCD and a limited range of substrates have been studied with the goal of trapping intermediates in sufficient yield for spectroscopic studies.…”

Crystal Structures of Fe 2+ Dioxygenase Superoxo, Alkylperoxo, and Bound Product Intermediates

“…Homoprotocatechuate 2,3-dioxygenase (2,3-HPCD) from Brevibacterium fuscum catalyzes the proximal extradiol ring cleavage of homoprotocatechuate (3,4-dihydroxyphenylacetate, HPCA) to form ring-opened 5-carboxymethyl-2-hydroxymuconic semialdehyde (13). The original structural study of 2,3-HPCD and its complex with HPCA employed a C-terminal deletion variant (14) to facilitate crystallization (10).…”

Exploring allosteric activation of LigAB from Sphingobium sp. strain SYK-6 through kinetics, mutagenesis and computational studies

“…From these studies it is clear that ring-cleaving dioxygenases, in general, have expansive substrate utilization profiles often being able to use 5 or more compounds as substrates in their respective dioxygenation reactions. Furthermore, two classes of inhibitors are described, those that function as chelators and oxidants that completely abolish dioxygenase activity by altering the nature of the iron cofactor [37][38][39][40][41][42][43][44][45][46][47][48], and organic substrate analogs which compete for binding to the active site [43,56]. In our previous work characterizing LigAB, we identified numerous substrates and inhibitors (Supplemental Table S2).…”