Abstract
Prodigiosin, a red linear tripyrrole pigment, is a typical secondary metabolite with numerous biological functions, such as anticancer, antibacterial and immunosuppressant activities, and is synthesized through a bifurcated biosynthesis pathway from 4-methoxy-2,2'-bipyrrole-5-carbaldehyde (MBC) and 2-methyl-3-n-amylpyrrole (MAP). The last step in the biosynthetic pathway of MBC is catalysed by PigF, which transfers a methyl group to 4-hydroxy-2,20-bipyrrole-5-carbaldehyde (HBC) to form the final product MBC. However, the catalytic mechanism of PigF is still elusive. In this study, crystal structures of apo PigF and S-adenosylhomocysteine (SAH)-bound PigF were determined. PigF forms a homodimer and each monomer consists of two domains: a C-terminal catalytic domain and an N-terminal dimerization domain. Apo PigF adopts an open conformation, while the structure of the complex with the product SAH adopts a closed conformation. The binding of SAH induces dramatic conformational changes of PigF, suggesting an induced-fit substrate-binding mechanism. Further structural comparison suggests that this induced-fit substrate-recognition mechanism may generally exist in O-methyltransferases. Docking and mutation studies identified three key residues (His98, His247 and Asp248) that are crucial for enzyme activity. The essential function of His247 and Asp248 and structure analysis suggests that both residues are involved in activation of the HBC substrate of PigF. The invariance of Asp248 in PigF further confirmed its essential role. The invariance and essential role of His98 in PigF suggests that it is involved in correctly positioning the substrate. This study provides new insight into the catalytic mechanism of PigF, reveals an induced-fit substrate-recognition model for PigF and broadens the understanding of O-methyltransferases.
Highlights
Serratia spp. belong to the large family Enterobacteriaceae and are widely distributed in different environments such as soil, water and plant surfaces; some strains of Serratia are opportunistic pathogens of humans, plants and some insects (Hejazi & Falkiner, 1997)
PigF is a member of the AdoMet-MTase superfamily and is essential for prodigiosin biosynthesis belonged to space group P1211 with unit-cell parameters a = 69.1, b = 52.36, c = 92.83 A, = 97.35 for native PigF and
We demonstrated that deletion of PigF results in the formation of an orange variant of prodigiosin (Fig. 3), which is consistent with the result observed in Serratia 39006 (Wilf & Salmond, 2012), indicating the formation of norprodigiosin
Summary
Serratia spp. belong to the large family Enterobacteriaceae and are widely distributed in different environments such as soil, water and plant surfaces; some strains of Serratia are opportunistic pathogens of humans, plants and some insects (Hejazi & Falkiner, 1997). The prodigiosin biosynthetic gene clusters from various Serratia strains have been cloned and sequenced (Harris et al, 2004) and typically include 14 genes. These gene clusters were functionally expressed in heterologous hosts such as Escherichia coli and Erwinia carotovora subsp. The biosynthesis of prodigiosin is proposed to take place via a bifurcated pathway in which 4-methoxy-2,20-bipyrrole-5-carbaldehyde (MBC) and 2-methyl-3-n-amylpyrrole (MAP) are separately synthesized before being condensed to form prodigiosin (Morrison, 1966; Wasserman et al, 1973; Harris et al, 2004; Williamson et al, 2005). PigF, which is a putative O-methyltransferase (Fig. 1), catalyses the final step in the biosynthetic pathway of MBC by transferring a methyl group to HBC to form the final product MBC (Williamson et al, 2005, 2006; Fig. 2).
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