Metal- and serine-dependent meta-cleavage product hydrolases utilize similar nucleophile-activation strategies


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 (kcat = 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 (Kd = 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. [on SciFinder(R)]

ACS Catal