Identification of functionally important residues and structural features in a bacterial lignostilbene dioxygenase

• Published in: The Journal of biological chemistry Vol. 294; no. 35; pp. 12911 - 12920
• Main Authors: Kuatsjah, Eugene, Verstraete, Meghan M., Kobylarz, Marek J., Liu, Alvin K.N., Murphy, Michael E.P., Eltis, Lindsay D.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 30-08-2019; American Society for Biochemistry and Molecular Biology
• Subjects: aromatic compound; bacterial catabolism; biomass conversion; carotenoid cleavage oxygenase; carotenoid dioxygenase; Crystallography, X-Ray; Dioxygenases - chemistry; Dioxygenases - metabolism; enzyme mechanism; Enzymology; lignin degradation; lignostilbene; lignostilbene dioxygenase (LSD); metalloenzyme; Models, Molecular; non-heme iron oxygenase; resveratrol; Sphingomonas - enzymology; X-ray crystallography; lignostilbene; non-heme iron oxygenase; bacterial catabolism; carotenoid cleavage oxygenase; metalloenzyme; X-ray crystallography; biomass conversion; carotenoid dioxygenase; lignin degradation; enzyme mechanism; resveratrol; lignostilbene dioxygenase (LSD); aromatic compound
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.RA119.009428

Lignostilbene-α,β-dioxygenase A (LsdA) from the bacterium Sphingomonas paucimobilis TMY1009 is a nonheme iron oxygenase that catalyzes the cleavage of lignostilbene, a compound arising in lignin transformation, to two vanillin molecules. To examine LsdA's substrate specificity, we heterologously produced the dimeric enzyme with the help of chaperones. When tested on several substituted stilbenes, LsdA exhibited the greatest specificity for lignostilbene (kcatapp = 1.00 ± 0.04 × 106m−1 s−1). These experiments further indicated that the substrate's 4-hydroxy moiety is required for catalysis and that this moiety cannot be replaced with a methoxy group. Phenylazophenol inhibited the LsdA-catalyzed cleavage of lignostilbene in a reversible, mixed fashion (Kic = 6 ± 1 μm, Kiu = 24 ± 4 μm). An X-ray crystal structure of LsdA at 2.3 Å resolution revealed a seven-bladed β-propeller fold with an iron cofactor coordinated by four histidines, in agreement with previous observations on related carotenoid cleavage oxygenases. We noted that residues at the dimer interface are also present in LsdB, another lignostilbene dioxygenase in S. paucimobilis TMY1009, rationalizing LsdA and LsdB homo- and heterodimerization in vivo. A structure of an LsdA·phenylazophenol complex identified Phe59, Tyr101, and Lys134 as contacting the 4-hydroxyphenyl moiety of the inhibitor. Phe59 and Tyr101 substitutions with His and Phe, respectively, reduced LsdA activity (kcatapp) ∼15- and 10-fold. The K134M variant did not detectably cleave lignostilbene, indicating that Lys134 plays a key catalytic role. This study expands our mechanistic understanding of LsdA and related stilbene-cleaving dioxygenases.