PqqE fromMethylobacterium extorquensAM1: a radicalS-adenosyl-l-methionine enzyme with an unusual tolerance to oxygen

Abstract

Methylobacterium extorquens AM1 is an aerobic facultative methylotroph known to secrete pyrroloquinoline quinone (PQQ), a cofactor of a number of bacterial dehydrogenases, into the culture medium. To elucidate the molecular mechanism of PQQ biosynthesis, we are focusing on PqqE which is believed to be the enzyme catalysing the first reaction of the pathway. PqqE belongs to the radical S-adenosyl-L-methionine (SAM) superfamily, in which most, if not all, enzymes are very sensitive to dissolved oxygen and rapidly inactivated under aerobic conditions. We here report that PqqE from M. extorquens AM1 is markedly oxygentolerant; it was efficiently expressed in Escherichia coli cells grown aerobically and affinity-purified to near homogeneity. The purified and reconstituted PqqE contained multiple (likely three) iron-sulphur clusters and showed the reductive SAM cleavage activity that was ascribed to the consensus 2+ cluster bound at the N-terminus region. Mo¨ssbauer spectrometric analyses of the as-purified and reconstituted enzymes revealed the presence of 2+ and [2Fe-2S] 2+clusters as the major forms with the former being predominant in the reconstituted enzyme. PqqE from M.extorquens AM1 may serve as a convenient tool for studying the molecular mechanism of PQQ biosynthesis, avoiding the necessity of establishing strictly anaerobic conditions.Keywords: Methylobacterium extorquens AM1/PQQ/ PqqE/radical SAM enzyme.Abbreviations: 5 0 dA, 5 0 -deoxyadenosine; HiPIPs, high-potential iron-sulphur proteins; IPTG, isopropyl--D-thiogalactopyranoside; PQQ, pyrroloquinoline quinone; SAM, S-adenosyl-L-methionine.Pyrroloquinoline quinone (PQQ) is an aromatic, tricyclic o-quinone that serves as a cofactor for a number of prokaryotic dehydrogenases, e.g. alcohol or glucose dehydrogenase (1, 2). The biosynthesis of PQQ is achieved in a series of reactions catalysed by enzymes encoded by genes located on pqq operon(s). The PQQ biosynthetic genes from several bacteria such as Acinetobacter calcoaceticus (3), Methylobacterium organophilum DSM 760 (4), Klebsiella pneumoniae (5), Pseudomonas fluorescens CHA0 (6) [reclassified as P.protegens CHA0 (7)], Methylobacterium extorquens AM1 (8), Enterobacter intermedium 60-2G (9) [reclassified as Kluyvera intermedia (10)] and Gluconobacter oxydans 621H (11) were reported and more than 125 bacterial species with PQQ biosynthetic capability were identified by bioinformatics analysis (12). Four to seven genes organized in operon(s) are responsible for PQQ biosynthesis in different bacteria (13). In K.pneumoniae, operon pqqABCDEF is involved in PQQ production. Among six genes, only pqqA, pqqC, pqqD and pqqE are exclusively required for PQQ production (14). Despite the fact that PQQ has been found decades ago, only little is known about its biosynthetic pathway. Although the putative function of each of the genes of pqq operon(s) has been proposed based on sequence analyses and homology modelling (15), the only biochemically confirmed function is that of pqqC, which encodes a protein catalysing o...

Demonstration That the Radical S-Adenosylmethionine (SAM) Enzyme PqqE Catalyzes de Novo Carbon-Carbon Cross-linking within a Peptide Substrate PqqA in the Presence of the Peptide Chaperone PqqD

“…2A). Taken together, this suggests that protein as-isolated contains a mixture of 2Fe-2S and 4Fe-4S clusters as recently reported by Saichana et al (23).…”

Nuclear Magnetic Resonance Structure and Binding Studies of PqqD, a Chaperone Required in the Biosynthesis of the Bacterial Dehydrogenase Cofactor Pyrroloquinoline Quinone

“…65 As well as the [4Fe-4S] cluster of the rSAM active site, the PqqE SPASM domain contains a [4Fe-4S] and a second cluster that may be a [2Fe-2S]. 65, 73 Electron paramagnetic resonance data indicate that the binding of PqqD affects at least one of the [4Fe-4S] clusters, which is consistent with the hypothesis that PqqD must bind in close proximity to the active site. 62 …”

Methods for Expression, Purification, and Characterization of PqqE, a Radical SAM Enzyme in the PQQ Biosynthetic Pathway

“…Mex PqqE, on the other hand, is much more tolerant to oxygen, and it can be expressed under aerobic conditions (Barr et al, 2016; Saichana et al, 2016), although it was initially expressed under anaerobic conditions (Latham et al, 2015). …”

“…Once the sequence is confirmed, the plasmid harboring the correct construct is cotransformed into E. coli BL21(DE3) (Stratagene) with vectors expressing the E. coli suf ABCDSE operon (pPH151) genes, which encodes proteins involved in Fe–S cluster repair and assembly (Nachin, Loiseau, Expert, & Barras, 2003). The vectors can also be cotransformed in E. coli Rosetta 2 (DE3) (Novagen), which contain rare codons that are seldom used in E. coli (Saichana et al, 2016). The pET vector contains a kanamycin (Kan) resistance gene, while the pPH151 is resistant to chloramphenicol (Cam); thus, the transformed strain is Kan and Cam resistant and can be grown in selective media with both antibiotics.…”

“…Purification of the K. pneumoniae His 6 tag-PqqE is performed under strict anaerobic conditions in an inert atmosphere glovebox (Wecksler et al, 2009), while Mex PqqE can be purified in anaerobic (Barr et al, 2016; Latham et al, 2015) or aerobic conditions (Saichana et al, 2016). The steps used in anaerobic and aerobic purifications are identical; the only difference being that aerobic purification is done in normal lab conditions, while anaerobic purification is done in a glovebox using degassed solutions and buffers.…”