Abstract
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 × 106 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 (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.
Highlights
Lignostilbene-␣,-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
Lignin-derived stilbenes are proposed to be produced in the bacterial catabolism of diaryl propane and phenylcoumarane [7, 12]
Coupled plasma MS (ICP-MS) analyses revealed that purified LsdA contained ϳ1 eq of iron per protomer and insignificant amounts of cadmium, cobalt, copper, zinc, manganese, nickel, and lead
Summary
Lignostilbene-␣,-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. When tested on several substituted stilbenes, LsdA exhibited the greatest specificity for lignostilbene (kcaaptp ؍1.00 ؎ 0.04 ؋ 106 M؊1 s؊1). Plants produce other stilbenes, such as resveratrol, arachidins, and astringins, as allelochemicals to protect against pathogens and physical damage [13] This variety of stilbenes may explain why some bacterial strains contain multiple LSD homologs [5, 7, 12, 14, 15]. The enzyme appears to act only on trans-stilbenes possessing a 4-hydroxy moiety [20]
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