Structural basis for methylphosphonate biosynthesis

Abstract

Methylphosphonate synthase (MPnS) produces methylphosphonate, a metabolic precursor to methane in the upper ocean. Here, we determine a 2.35-angstrom resolution structure of MPnS and discover that it has an unusual 2-histidine-1-glutamine iron-coordinating triad. We further solve the structure of a related enzyme, hydroxyethylphosphonate dioxygenase from Streptomyces albus (SaHEPD), and find that it displays the same motif. SaHEPD can be converted into an MPnS by mutation of glutamine-adjacent residues, identifying the molecular requirements for methylphosphonate synthesis. Using these sequence markers, we find numerous putative MPnSs in marine microbiomes and confirm that MPnS is present in the abundant Pelagibacter ubique. The ubiquity of MPnS-containing microbes supports the proposal that methylphosphonate is a source of methane in the upper, aerobic ocean, where phosphorus-starved microbes catabolize methylphosphonate for its phosphorus.

Figure 1

The reactions of MPnS and HEPD have been proposed to proceed through a common radical intermediate. The hydrogen derived from the C2 pro-R position in 2-HEP is highlighted red throughout. The radical recombination in MPnS or HEPD (17) is colored red or blue, respectively. For an alternative mechanism involving a cationic intermediate, see Fig. S9.

Figure 2

Active site structure of NmMPnS and SaHEPD. (A) The NmMPnS active site is composed of residues from the β1 domain (cyan) and the α1′ domain (pink), with two key residues discussed in the text in light brown. Fe(II) (orange sphere) is ligated by substrate, Gln152, His148, and His190. Although electron density cannot be used to distinguish oxygen from nitrogen, we are showing the oxygen of the Gln side chain coordinating Fe(II). (B) SaHEPD active site composed of residues from the β1 domain (yellow) and the α1′ domain (green) for two protomers of the asymmetric unit in which Gln153 is in the iron-binding conformation. (C) Alternative conformation of SaHEPD, where Gln153 is displaced from the Fe(II).

Figure 3

Proposed intermediates for MPnS and SaHEPD. (A) Gln152 in MPnS coordinates the formate intermediate, enabling H-atom abstraction by the MPn radical. (B) Formate displaces Gln153 from its iron-coordinating position in SaHEPD, preventing H-atom abstraction by the MPn-based radical and promoting reaction with the iron-bound hydroxide.

Figure 4

Phylogeny of MPnS, HEPD, and HppE-like proteins. HEPD proteins are colored based on the iron-ligating facial triad: class I HEPD, 2-His-1-Glu (green); or class II HEPD, 2-His-1-Gln (brown). MPnS proteins are colored based on the amino acid at position 184, either Ile (blue) or Val (magenta). Complete labeling of sequence names is provided in Fig. S10.