Metal- and Serine-Dependent Meta-Cleavage Product Hydrolases Utilize Similar Nucleophile-Activation StrategiesClick to copy article linkArticle link copied!
- Eugene KuatsjahEugene KuatsjahGenome Science and Technology Program, Life Sciences Institute, The University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3More by Eugene Kuatsjah
- Anson C. K. ChanAnson C. K. ChanDepartment of Microbiology and Immunology, The University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3More by Anson C. K. Chan
- Timothy E. HurstTimothy E. HurstDepartment of Chemistry, Queen’s University, Kingston, Ontario, Canada K7L 3N6More by Timothy E. Hurst
- Victor SnieckusVictor SnieckusDepartment of Chemistry, Queen’s University, Kingston, Ontario, Canada K7L 3N6More by Victor Snieckus
- Michael E. P. MurphyMichael E. P. MurphyDepartment of Microbiology and Immunology, The University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3More by Michael E. P. Murphy
- Lindsay D. Eltis*Lindsay D. Eltis*E-mail [email protected]Department of Microbiology and Immunology and Department of Biochemistry and Molecular Biology, The University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3More by Lindsay D. Eltis
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 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.
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