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

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 (k(cat)(app) = 1.00 ± 0.04 × 10(6) m(−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 (K(ic) = 6 ± 1 μm, K(iu) = 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 Phe(59), Tyr(101), and Lys(134) as contacting the 4-hydroxyphenyl moiety of the inhibitor. Phe(59) and Tyr(101) substitutions with His and Phe, respectively, reduced LsdA activity (k(cat)(app)) ∼15- and 10-fold. The K134M variant did not detectably cleave lignostilbene, indicating that Lys(134) plays a key catalytic role. This study expands our mechanistic understanding of LsdA and related stilbene-cleaving dioxygenases.