Robert M. Cicchillo

Synthesis of methylphosphonic acid by marine microbes: A source for methane in the aerobic ocean

Ocean Methane Is methylphosphonate a natural product? If it is, then its catabolism by microrganisms seeking phosphorus might explain the ubiquity of methane in the oceans. Anaerobic Archaea, so far, are the only significant source of methane known. Thus, Metcalf et al. (p. 1104) looked for evidence of methylphosphonate biochemistry in the widespread archaeon Nitrosopumilus maritimus. The organism can synthesize C-P bonds using phosphoenolpyruvate mutase. By using its gene, ppm, as a marker, a possible phosphonate biosynthesis gene cluster was identified, and adjacent to this a gene for a cupin was found. Together with Fe(II) and O2 the cupin catalyzed the production of methylphosphonate and formate. Homologs of the cupin gene are found in a wide range of common marine microbes, including Pelagibacter ubique and Prochlorococcus. The archaeon Nitrosopumilus maritimus makes what may be a major source of ocean methane. Relative to the atmosphere, much of the aerobic ocean is supersaturated with methane; however, the source of this important greenhouse gas remains enigmatic. Catabolism of methylphosphonic acid by phosphorus-starved marine microbes, with concomitant release of methane, has been suggested to explain this phenomenon, yet methylphosphonate is not a known natural product, nor has it been detected in natural systems. Further, its synthesis from known natural products would require unknown biochemistry. Here we show that the marine archaeon Nitrosopumilus maritimus encodes a pathway for methylphosphonate biosynthesis and that it produces cell-associated methylphosphonate esters. The abundance of a key gene in this pathway in metagenomic data sets suggests that methylphosphonate biosynthesis is relatively common in marine microbes, providing a plausible explanation for the methane paradox.

An unusual carbon-carbon bond cleavage reaction during phosphinothricin biosynthesis

Natural products containing phosphorus–carbon bonds have found widespread use in medicine and agriculture. One such compound, phosphinothricin tripeptide, contains the unusual amino acid phosphinothricin attached to two alanine residues. Synthetic phosphinothricin (glufosinate) is a component of two top-selling herbicides (Basta and Liberty), and is widely used with resistant transgenic crops including corn, cotton and canola. Recent genetic and biochemical studies showed that during phosphinothricin tripeptide biosynthesis 2-hydroxyethylphosphonate (HEP) is converted to hydroxymethylphosphonate (HMP). Here we report the in vitro reconstitution of this unprecedented C(sp3)–C(sp3) bond cleavage reaction and X-ray crystal structures of the enzyme. The protein is a mononuclear non-haem iron(ii)-dependent dioxygenase that converts HEP to HMP and formate. In contrast to most other members of this family, the oxidative consumption of HEP does not require additional cofactors or the input of exogenous electrons. The current study expands the scope of reactions catalysed by the 2-His–1-carboxylate mononuclear non-haem iron family of enzymes.

Hydroperoxylation by hydroxyethylphosphonate dioxygenase.

Hydroxyethylphosphonate dioxygenase (HEPD) catalyzes the O2-dependent cleavage of the carbon−carbon bond of 2-hydroxyethylphosphonate (2-HEP) to afford hydroxymethylphosphonate (HMP) and formate without input of electrons or use of any organic cofactors. Two mechanisms have been proposed to account for this reaction. One involves initial hydroxylation of substrate to an acetal intermediate and its subsequent attack onto an Fe(IV)-oxo species. The second mechanism features initial hydroperoxylation of substrate followed by a Criegee rearrangement. To distinguish between the two mechanisms, substrate analogues were synthesized and presented to the enzyme. Hydroxymethylphosphonate was converted into phosphate and formate, and 1-hydroxyethylphosphonate was converted to acetylphosphate, which is an inhibitor of the enzyme. These results provide strong support for a Criegee rearrangement with a phosphorus-based migrating group and require that the O−O bond of molecular oxygen is not cleaved prior to substrate activation. (2R)-Hydroxypropylphosphonate partitioned between conversion to 2-oxopropylphosphonate and hydroxymethylphosphonate, with the latter in turn converted to phosphate and formate. Collectively, these results support a mechanism that proceeds by hydroperoxylation followed by a Criegee rearrangement.

On the Stereochemistry of 2-Hydroxyethylphosphonate Dioxygenase

Stereochemical investigations have shown that the conversion of 2-hydroxyethylphosphonate to hydroxymethylphosphonate by the enzyme HEPD involves removal of the pro-S hydrogen at C2 and, surprisingly, the loss of stereochemical information at C1. As a result, the mechanisms previously proposed for HEPD must be re-evaluated.

Mechanism and Substrate Recognition of 2-Hydroxyethylphosphonate Dioxygenase

HEPD belongs to the superfamily of 2-His-1-carboxylate non-heme iron-dependent dioxygenases. It converts 2-hydroxyethylphosphonate (2-HEP) to hydroxymethylphosphonate (HMP) and formate. Previously postulated mechanisms for the reaction catalyzed by HEPD cannot explain its conversion of 1-HEP to acetylphosphate. Alternative mechanisms that involve either phosphite or methylphosphonate as intermediates, which potentially explain all experimental studies including isotope labeling experiments and use of substrate analogues, were investigated. The results of these studies reveal that these alternative mechanisms are not correct. Site-directed mutagenesis studies of Lys16, Arg90, and Tyr98 support roles of these residues in binding of 2-HEP. Mutation of Lys16 to Ala resulted in an inactive enzyme, whereas mutation of Arg90 to Ala or Tyr98 to Phe greatly decreased kcat/Km,2-HEP. Furthermore, the latter mutants could not be saturated in O2. These results suggest that proper binding of 2-HEP is important for O2 activation and that the enzyme uses a compulsory binding order with 2-HEP binding before O2. The Y98F mutant produces methylphosphonate as a minor side product providing indirect support for the proposal that the last step during catalysis involves a ferric hydroxide reacting with a methylphosphonate radical.