Strain SYK-6 of the bacterium sp. catabolizes lignin-derived biphenyl via a -cleavage pathway. In this pathway， LigY is proposed to catalyze the hydrolysis of the -cleavage product (MCP) 4，11-dicarboxy-8-hydroxy-9-methoxy-2-hydroxy-6-oxo-6-phenyl-hexa-2，4-dienoate. Here， we validated this reaction by identifying 5-carboxyvanillate and 4-carboxy-2-hydroxypenta-2，4-dienoate as the products and determined the and / values as 9.3 ± 0.6 s and 2.5 ± 0.2 × 10 m s， respectively. Sequence analyses and a 1.9 Å resolution crystal structure established that LigY belongs to the amidohydrolase superfamily， unlike previously characterized MCP hydrolases， which are serine-dependent enzymes of the α/β-hy... More
Strain SYK-6 of the bacterium sp. catabolizes lignin-derived biphenyl via a -cleavage pathway. In this pathway， LigY is proposed to catalyze the hydrolysis of the -cleavage product (MCP) 4，11-dicarboxy-8-hydroxy-9-methoxy-2-hydroxy-6-oxo-6-phenyl-hexa-2，4-dienoate. Here， we validated this reaction by identifying 5-carboxyvanillate and 4-carboxy-2-hydroxypenta-2，4-dienoate as the products and determined the and / values as 9.3 ± 0.6 s and 2.5 ± 0.2 × 10 m s， respectively. Sequence analyses and a 1.9 Å resolution crystal structure established that LigY belongs to the amidohydrolase superfamily， unlike previously characterized MCP hydrolases， which are serine-dependent enzymes of the α/β-hydrolase superfamily. The active-site architecture of LigY resembled that of α-amino-β-carboxymuconic-ϵ-semialdehyde decarboxylase， a class III amidohydrolase， with a single zinc ion coordinated by His-6， His-8， His-179， and Glu-282. Interestingly， we found that LigY lacks the acidic residue proposed to activate water for hydrolysis in other class III amidohydrolases. Moreover， substitution of His-223， a conserved residue proposed to activate water in other amidohydrolases， reduced the to a much lesser extent than what has been reported for other amidohydrolases， suggesting that His-223 has a different role in LigY. Substitution of Arg-72， Tyr-190， Arg-234， or Glu-282 reduced LigY activity over 100-fold. On the basis of these results， we propose a catalytic mechanism involving substrate tautomerization， substrate-assisted activation of water for hydrolysis， and formation of a -diol intermediate. This last step diverges from what occurs in serine-dependent MCP hydrolases. This study provides insight into C-C-hydrolyzing enzymes and expands the known range of reactions catalyzed by the amidohydrolase superfamily.
