UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Characterization of metalloenzymes involved in the bacterial catabolism of lignin-derived biaryls Kuatsjah, Eugene


Bacteria play a central role in degrading aromatic compounds, including those derived from lignin, a heterogeneous aromatic polymer and a major component of woody biomass. These catabolic pathways and enzymes play a critical role in the global carbon cycle and have biocatalytic potential in the valorization of biomass. Nevertheless, many of these pathways and enzymes remain poorly understood. This thesis describes the characterization of three such catabolic enzymes, LsdA, LigZ and LigY, each of which has a metal cofactor. LsdA from Sphingomonas paucimobilis TMY1009 is an Fe²⁺-dependent oxygenase that catalyzes the cleavage of stilbenoids into aldehydes. In kinetic anal-yses, LsdA only transformed 4-hydroxystilbenes and was inhibited by phenylazophenol. Crystallo-graphic and mutagenesis analyses established that Lys-134 is essential for catalysis, consistent with a role in deprotonating the stilbene’s 4-hydroxyl. LigZ and LigY catalyze successive reactions in 2,2’-dihydroxy-3,3’-dimethoxy-5,5’-dicarboxy-biphenyl (DDVA) catabolism by Sphingobium sp. SYK-6. In this study, highly active preparations of LigZ, an Fe²⁺-dependent extradiol dioxygenase, afforded an accurate description of the enzyme’s substrate specificity and mechanism-based inactivation. 4,11-Dicarboxy-8-hydroxy-9-methoxy-2-hydroxy-6-oxo-6-phenyl-hexa-2,4-dienoate (DCHM-HOPDA) was identified as the meta-cleavage product (MCP) of the LigZ reaction and was shown to undergo a rapid, reversible non-enzymatic transformation under physiological conditions (t½ ~5 min). The enolate form of the MCP is the substrate of LigY, which catalyzed the hydrolysis of DCHM-HOPDA to 5-carboxyvanillate (5CVA) and 4-carboxy-2-hydroxypenta-2,4-dienoate (CHPD). Phy-logenetic, biochemical and structural analyses identified LigY as a Zn²⁺-dependent amidohydrolase, in contrast to all known MCP hydrolases, which are serine-dependent enzymes. Nevertheless, transient-state kinetic analyses revealed a catalytic intermediate, ESred, with a red-shifted spectrum similar to that observed in serine-dependent MCP hydrolases. Further, 4-methyl HOPDA inhibited LigY and yielded a complex with a spectrum similar to that of ESred. Crystallographic analyses of this complex revealed the MCP binds the Zn²⁺ in a bidentate manner. Together, the data support a mechanism in which the nucleophile is activated in the same substrate-assisted manner as in the serine-dependent enzymes, but that in LigY, the nucleophile is water, such that C-C fission is preceded by a gem-diol intermediate. Overall, this thesis provides important insights into several lignin-degrading enzymes and the superfamilies to which they belong.

Item Media

Item Citations and Data


Attribution-NonCommercial-NoDerivatives 4.0 International