Structure and function of a flavin-dependent S-monooxygenase from garlic (Allium sativum).

Allicin is a component of the characteristic smell and flavor of garlic (Allium sativum). A flavin-containing monooxygenase (FMO) produced by A. sativum (AsFMO) was previously proposed to oxidize S-allyl-l-cysteine (SAC) to alliin, an allicin precursor. Here, we present a kinetic and structural characterization of AsFMO that suggests a possible contradiction to this proposal. Results of steady-state kinetic analyses revealed that AsFMO exhibited negligible activity with SAC; however, the enzyme was highly active with l-cysteine, N-acetyl-l-cysteine, and allyl mercaptan. We found that allyl mercaptan with NADPH was the preferred substrate-cofactor combination. Rapid-reaction kinetic analyses showed that NADPH binds tightly (KD of ∼2 µm) to AsFMO and that the hydride transfer occurs with pro-R stereospecificity. We detected the formation of a long-wavelength band when AsFMO was reduced by NADPH, probably representing the formation of a charge-transfer complex. In the absence of substrate, the reduced enzyme, in complex with NADP+, reacted with oxygen and formed an intermediate with a spectrum characteristic of C4a-hydroperoxyflavin, which decays several orders of magnitude more slowly than the kcat The presence of substrate enhanced C4a-hydroperoxyflavin formation and, upon hydroxylation, oxidation occurred with a rate constant similar to the kcat The structure of AsFMO complexed with FAD at 2.08-Å resolution features two domains for binding of FAD and NADPH, representative of class B flavin monooxygenases. These biochemical and structural results are consistent with AsFMO being an S-monooxygenase involved in allicin biosynthesis through direct formation of sulfenic acid and not SAC oxidation.

Crystallization of Phi29 spindle-shaped nano-bar anti-receptor with glycosidase domain.

Bacteriophage phi29 is a small, well-characterized dsDNA virus that infects Bacillus subtilis. The anti-receptor of phi29 consists of oligomers of the 854-residue protein gp12 and plays an essential role in infection initiation by binding to the receptor on the host cell surface. Oligomers of gp12 exhibit a narrow spindle-shaped configuration 15 nm in length as revealed by electron microscopy and thus are potentially useful nanoscale tools, building blocks, or motor arms. To understand the mechanism of viral infection initiation and to provide a basis for engineering recombinant gp12 for nanotechnology applications, we have initiated structural and bioinformatics studies of gp12. We report here the growth of crystals of gp12 that diffract to 3.0 A resolution. The space group is P3(1)21 or P3(2)21 with unit cell lengths of a = 84.4 A and c = 167.6 A. The asymmetric unit is predicted to contain one gp12 molecule and 32% solvent (VM = 1.8 A3/Da). Domain boundary analysis revealed that gp12 may harbor three domains besides a 24 residue auto-cleave region. The N-terminal half of gp12 contains a domain with about 400 residues that held 44% sequence identity to endopolygalacturonase, a fungal glycosyl hydrolase that catalyzes hydrolysis of the polygalacturonic acid alpha1-4 glycosidic linkage found in plant cell walls. Interestingly, the cell wall of Bacillus subtilis contains a polysaccharide component made from two sugar monomers, N-acetylmuramic acid and N-acetylglucosamine, which resemble alpha-galacturonic acid in that they possess a six-membered pyranose ring. Hence, polygalacturonic acid of plant cell walls and peptidoglycan of bacterial cell walls may offer a similar topography in relation to the polysaccharides. These results suggest a function for gp12 as a cell-wall degrading enzyme in addition to its role in recognition of the host receptor.