The bacterial meta-cleavage hydrolase LigY belongs to the amidohydrolase superfamily, not to the α/β-hydrolase superfamily

Abstract

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.

Bacterial catabolism of lignin‐derived aromatics: New findings in a recent decade: Update on bacterial lignin catabolism

“…In addition to the catabolic pathways, a large number of detailed biochemical studies and structural analyses on catabolic enzymes, including ring‐cleavage extradiol dioxygenases, protocatechuate 4,5‐cleavage pathway enzymes and hydrolase and decarboxylase involved in biphenyl catabolism, were performed (Hobbs et al ., ; Barry and Taylor, ; Sugimoto et al ., ; Barry et al ., ; Zhang et al ., ; Vladimirova et al ., ; Kuatsjah et al ., ). Here, we cover two topics regarding ring‐cleavage extradiol dioxygenase.…”

The Pyruvate Aldol Condensation Product: A Metabolite that Escaped Synthetic Preparation for Over a Century

“…5 Only recent evidence of its occurrence in the 5,5'dehydrodivanillate (DDVA) pathway had been reported. 6,7 The first study 6 reported an enzymatic production of this compound from its aldol product precursor, 4-hydroxy-4methyl-2-oxoglutaric acid (HMOG), with analysis by HPLC but without verification with an authentic analytical standard. The more recent work 7 also reported evidence of CHPD based on the enzymatic fragmentation of an upstream lignin derived aromatic compound that lead to the suspected CHPD and 5-carboxyvanillate.…”

Bacterial Catabolism of Biphenyls: Synthesis and Evaluation of Analogues

“…BphD is a serine‐dependent member of the α/β‐hydrolase superfamily, typical of all but one of the MCP hydrolases characterized to date. The only MCP hydrolase that differs is LigY, which is a Zn 2+ ‐dependent member of the amidohydrolase superfamily . It is unclear how these two enzymes use such different catalytic machinery to catalyze similar reactions.…”