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 (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 consist...
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