Structural Properties and Catalytic Implications of the SPASM Domain Iron–Sulfur Clusters in Methylorubrum extorquens PqqE

Abstract

Understanding the relationship between the metallocofactor and its protein environment is the key to uncovering the mechanism of metalloenzymes. PqqE, a radical S-adenosylmethionine enzyme in pyrroloquinoline quinone (PQQ) biosynthesis, contains three iron−sulfur cluster binding sites. Two auxiliary iron−sulfur cluster binding sites, designated as AuxI and AuxII, use distinctive ligands compared to other proteins in the family while their functions remain unclear. Here, we investigate the electronic properties of these iron−sulfur clusters and compare the catalytic efficiency of wild-type (WT) Methylorubrum extorquens AM1 PqqE to a range of mutated constructs. Using native mass spectrometry, protein film electrochemistry, and electron paramagnetic resonance spectroscopy, we confirm the previously proposed incorporation of a mixture of [2Fe-2S] and [4Fe-4S] clusters at the AuxI site and are able to assign redox potentials to each of the three iron− sulfur clusters. Significantly, a conservative mutation at AuxI, C268H, shown to selectively incorporate a [4Fe-4S] cluster, catalyzes an enhancement of uncoupled S-adenosylmethionine cleavage relative to WT, together with the elimination of detectable peptide cross-linked product. While a [4Fe-4S] cluster can be tolerated at the AuxI site, the aggregate findings suggest a functional [2Fe-2S] configuration within the AuxI site. PqqE variants with nondestructive ligand replacements at AuxII also show that the reduction potential at this site can be manipulated by changing the electronegativity of the unique aspartate ligand. A number of novel mechanistic features are proposed based on the kinetic and spectroscopic data. Additionally, bioinformatic analyses suggest that the unique ligand environment of PqqE may be relevant to its role in PQQ biosynthesis within an oxygen-dependent biosynthetic pathway.

Mechanism of Reduction of an Aminyl Radical Intermediate in the Radical SAM GTP 3′,8-Cyclase MoaA

“…Elucidation of the catalytic function of auxiliary clusters in radical SAM enzymes in the SPASM-twitch family remains difficult. While reduction potentials of several other members of the SPASM-twitch family have been reported, ,,, ambiguity remains about their functions in catalysis due to the lack of understanding in the reduction potentials of reaction intermediates and the redox state of the auxiliary clusters during catalytic turnover. In this study, we used a combination of PFV, EPR, and DFT calculation to provide experimental evidence that the reduced 1+ state auxiliary cluster of MoaA reduces the aminyl radical intermediate to form the final product.…”

Radical SAM Enzymes and Ribosomally‐Synthesized and Post‐translationally Modified Peptides: A Growing Importance in the Microbiomes

“…Like the ranthisynthase CteB, these enzymes share the canonical (β/α) 6 TIM barrel fold but differ in the nature of auxiliary iron–sulfur clusters. Whereas SuiB harbors in its C -terminal part a SPASM domain characterized by two auxiliary [4Fe-4S] clusters, it has been shown that PqqE contains one [2Fe-2S] and one [4Fe-4S] auxiliary clusters, with possible conversion of the [2Fe-2S] into a [4Fe-4S] cluster ( Zhu et al, 2020 ). In addition, one of the auxiliary clusters of PqqE is not coordinated by four cysteine residues, but by three cysteine residues and a conserved aspartate which likely modulates its redox properties ( Barr et al, 2018 ; Tao et al, 2019 ).…”

Current Advancements in Sactipeptide Natural Products

“…Structural data shows that SkfB has an N-terminal RiPP recognition element (RRE) domain for peptide-binding. Subsequent is a classic partial (β/α) 6 barrel structure bounding the [4Fe-4S] cluster, and the C-terminal is a twitch domain harboring a [2Fe-2S] cluster, which is the same with the pyrroloquinoline quinone biosynthesis enzyme PqqE (Grell et al, 2018 ; Zhu et al, 2020 ). Unambiguously, SkfB catalyzes hydrogen abstraction from the α-carbon of Met12 of precursor peptide SkfA (Bruender and Bandarian, 2016 ).…”