Metal- and Serine-Dependent Meta-Cleavage Product Hydrolases Utilize Similar Nucleophile-Activation Strategies

Abstract

LigY catalyzes the hydrolysis of a meta-cleavage product (MCP), 4,11-dicarboxy-8-hydroxy-9-methoxy-2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (DCHM-HOPDA), in the bacterial catabolism of lignin-derived biphenyl. Most characterized MCP hydrolases are serine-dependent, with hydrolysis proceeding via enol–keto tautomerization followed by an acyl-enzyme intermediate. In contrast, LigY is Zn2+-dependent, with hydrolysis proposed to proceed via tautomerization followed by formation of a gem-diol intermediate. Transient-state kinetic analysis of DCHM-HOPDA turnover revealed the formation of an intermediate possessing a bathochromically shifted spectrum (λmax = 508 nm), similar to that of the ESred intermediate observed during tautomerization in serine-dependent hydrolases. Neither the formation (1/τ1 ≈ 137 s–1) nor the decay (1/τ2 ≈ 23 s–1) of ESred was rate-limiting (k cat = 9.7 ± 0.3 s–1). Furthermore, the rate of ESred decay was 3.4-fold slower in deuterated buffer, suggesting a proton-transfer reaction consistent with substrate ketonization. LigY turned over 4-carboxy-HOPDA but not 4-methyl-HOPDA, suggesting that the carboxylate is essential for catalysis. Titration of LigY with 4-methyl-HOPDA yielded a species with a spectrum similar to that of ESred (K d = 25 ± 1 μM). A 2.4-Å crystal structure of the LigY·4-methyl-HOPDA complex, which also had a spectrum like ESred, revealed the ligand coordinated to the Zn2+ in a bidentate manner via the 1-carboxylate and 2-oxo groups. Overall, the data support a mechanism in which the metallocenter primarily catalyzes substrate tautomerization and the water required for the hydrolytic half-reaction is activated in a substrate-assisted manner. This study provides insight into C–C bond hydrolases as well as the versatility of the catalytic machinery of metallohydrolases.

Kj-mhpC Enzyme in Klebsiella jilinsis 2N3 Is Involved in the Degradation of Chlorimuron-Ethyl via De-Esterification

“…It is reported that 2-hydroxy −6-oxotriene diacid hydroxide (mhpC) can hydrolyze C5−C6 of 2-hydroxy-6-keto-nona-2,4-diene1,9-dioic acid to obtain succinic acid and 2-hydroxy-2,4-dienoic acid. 18 In addition, the immobilization of K. jilinsis strain 2N3 with corn straw biochar is helpful to promote the degradation of nicosulfuron in soil; improve the activities of urease, sucrase, dehydrogenase, and catalase; and restore the function and composition of soil microorganisms. 19 Moreover, our previous study reported that the Kj-mhpC gene in K. jilinsis 2N3 is involved in the degradation of chlorimuron-ethyl.…”

“…The hydrolysis and cleavage of carbon–carbon bonds occur via an enzymatic process, which involves the key steps of the bacterial degradation pathway of organic pollutants. It is reported that 2-hydroxy −6-oxotriene diacid hydroxide (mhpC) can hydrolyze C5–C6 of 2-hydroxy-6-keto-nona-2,4-diene1,9-dioic acid to obtain succinic acid and 2-hydroxy-2,4-dienoic acid …”

Biomimetic single Al-OH site with high acetylcholinesterase-like activity and self-defense ability for neuroprotection

“…First, the nucleophilic hydroxyl group (–OH) of serine attacks the carbon (phosphorus) atoms of substrates to form the intermediates. Meanwhile, the proximal basic imidazole groups and Lewis acid Zn 2+ sites respectively enrich the electron density of nucleophilic groups and improve the accessibility of carbon (phosphorus) atoms 15 , 16 . Subsequently, the dissociation of the ester bond occurs and generates the first product with the assistance of protonation 17 , 18 .…”