PhnJ – A novel radical SAM enzyme from the C–P lyase complex

Abstract

PhnJ from the C-P lyase complex catalyzes the cleavage of the carbon-phosphorus bond in ribose-1-phosphonate-5-phosphate (PRPn) to produce methane and ribose-1,2-cyclic-phosphate-5-phosphate (PRcP). This protein is a novel radical SAM enzyme that uses glycyl and thiyl radicals as reactive intermediates in the proposed reaction mechanism. The overall reaction is initiated with the reductive cleavage of S-adenosylmethionine (SAM) by a reduced [4Fe-4S] 1 + -cluster to form an Ado-CH 2 • radical intermediate. This intermediate abstracts the proR hydrogen from Gly-32 of PhnJ to form Ado-CH 3 and a glycyl radical. In the next step, there is hydrogen atom transfer from Cys-272 to the Gly-32 radical to generate a thiyl radical. The thiyl radical attacks the phosphorus center of the substrate, PRPn, to form a transient thiophosphonate radical intermediate. This intermediate collapses via homolytic C-P bond cleavage and hydrogen atom transfer from the proS hydrogen of Gly-32 to produce a thiophosphate intermediate, methane, and a radical intermediate at Gly-32. The final product, PRcP, is formed by nucleophilic attack of the C2-hydroxyl on the transient thiophosphate intermediate. This reaction regenerates the free thiol group of Cys-272. After hydrogen atom transfer from Cys-272 to the Gly-32 radical, the entire process is repeated with another substrate molecule without the use of another molecule of SAM or involvement from the [4Fe-4S]-cluster again.

Mechanism of a Class C Radical S-Adenosyl-l-methionine Thiazole Methyl Transferase

“…On the basis of literature precedent, an example of the latter could be the α-hydrogens of Gly. 43 , 44 Lastly, the hydrogen could originate from the methyl group of 5′-dA (pathway c ).…”

“…This behavior has previously been observed, such as the transfer of Cα hydrogens from a Gly residue. 43 , 44 In principle, hydrogen transfer could also originate from other amino acids or very tightly hydrogen bonded positions that would only exchange upon reaction. Because TbtI contains several conserved Gly residues ( Figure S2 ), we reacted a 20-fold excess of substrate 1 and CD 3 -SAM with TbtI in buffered D 2 O and analyzed the product by ESI-MS ( Figure S9 ).…”

MBPpred: Proteome-wide detection of membrane lipid-binding proteins using profile Hidden Markov Models

“…The other zinc finger domains are also associated with binding, but of other substrates like DNA and RNA. Two very interesting cases were those of PhnJ and ACBP, both domains mainly found in prokaryotes [56,57]. PhnJ is a phosphonate utilization domain [58], and even though this process is not relevant to membrane binding directly, it has been shown that phosphonate can act as an inhibitor to phosphate binding [59], showing that this distant relationship found in this case could help explain the divergence of function in related domains between prokaryotes and eukaryotes.…”

MBPpred: Proteome-wide detection of membrane lipid-binding proteins using profile Hidden Markov Models

“…The other zinc finger domains are also associated with binding, but of other substrates like DNA and RNA. Two very interesting cases were those of PhnJ and ACBP, both domains mainly found in prokaryotes [56,57]. PhnJ is a phosphonate utilization domain [58], and even though this process is not relevant to membrane binding directly, it has been shown that phosphonate can act as an inhibitor to phosphate binding [59], showing that this distant relationship found in this case could help explain the divergence of function in related domains between prokaryotes and eukaryotes.…”