O2-independent demethylation of trimethylamine N-oxide by Tdm of Methylocella silvestris

Bacterial trimethylamine N-oxide (TMAO) demethylase, Tdm, carries out an unusual oxygen-independent demethylation reaction, resulting in the formation of dimethylamine and formaldehyde. In this study, sitedirected mutagenesis, homology modelling and metal analyses by inorganic mass spectrometry have been applied to gain insight into metal stoichiometry and underlying catalytic mechanism of Tdm of Methylocella silvestris BL2. Herein, we demonstrate that active Tdm has 1 molar equivalent of Zn2+ and 1 molar equivalent of non-heme Fe2+. We further investigated Zn2+ and Fe2+-binding sites through homology modelling and sitedirected mutagenesis and found that Zn2+ is coordinated by a 3-sulfur-1-O motif. An aspartate residue (D198) likely bridges Fe2+ and Zn2+ centres, either directly or indirectly via H-bonding through a neighbouring H2O molecule. H276 contributes to Fe2+ binding, mutation of which results in an inactive enzyme, and the loss of iron, but not zinc. Site-directed mutagenesis of Tdm also led to the identification of three hydrophobic aromatic residues likely involved in substrate coordination (F259, Y305, W321), potentially through a cation- interaction. Furthermore, a cross-over experiment using a substrate analogue gave direct evidence that a trimethylamine-alike intermediate was produced during the Tdm catalytic cycle, suggesting TMAO has a dual role of being both a substrate and an oxygen donor for formaldehyde formation. Together, our results provide novel insight into the role of Zn2+ and Fe2+ in the catalysis of TMAO demethylation by this unique oxygenindependent enzyme

A single sensor controls large variations in zinc quotas in a marine cyanobacterium

Marine cyanobacteria are critical players in global nutrient cycles that crucially depend on trace metals in metalloenzymes, including zinc for CO2 fixation and phosphorus acquisition. How strains proliferating in the vast oligotrophic ocean gyres thrive at ultra-low zinc concentrations is currently unknown. Using Synechococcus sp. WH8102 as a model we show that its zinc-sensor protein Zur differs from all other known bacterial Zur proteins in overall structure and the location of its sensory zinc site. Uniquely, Synechococcus Zur activates metallothionein gene expression, which supports cellular zinc quotas spanning two orders of magnitude. Thus, a single zinc sensor facilitates growth across pico- to micromolar zinc concentrations with the bonus of banking this precious resource. The resultant ability to grow well at both ultra-low and excess zinc, together with overall lower zinc requirements, likely contribute to the broad ecological distribution of Synechococcus across the global oceans

Zinc on the move : insights towards understanding zinc homeostasis in the open ocean cyanobacterium Synechococcus sp. WH8102.

Since the discovery of zinc as an essential element for living organisms including humans, animals, plants, fungi and bacteria, much has been learnt about zinc in biological systems, ranging from its effects at the whole organism level, including the uptake/efflux of zinc, identification of important zinc-regulator proteins, down to structural, thermodynamic, and kinetic details of zinc-protein interactions. One of the most exciting fields to work on, currently, is understanding the importance of zinc “on the move” - in particular the “acquisition and homeostasis” of this micronutrient element by the organisms. In this wider context, zinc homeostasis by open ocean cyanobacteria, which occupy variable ecosystems with an erratic nutrient supply, has become of recent interest. In an attempt to resolve how open ocean cyanobacteria persist in regions where the zinc concentration is thought to be limited, we hypothesised that the cyanobacterium Synechococcus sp. WH8102 might provide an extracellular zinc scavenger for the acquisition of this essential element. Therefore, the current study developed a method to isolate and purify the putative biogenic zinc-binding ligand (zincophore) using polystyrene-divinylbenzene resin and liquid chromatographies. However, it seems likely Synechococcus sp. WH8102 produce ligands that can bind to zinc only under zinc-depleted conditions. Then, we performed zinc limitation and repletion experiments on axenic cultures of marine Synechococcus sp. WH8102, and data determined that this strain mounted an adaptive response for zinc under depleted and replete conditions, resulting in the induction and/or repression of a number of proteins. As a homologue to the Fur family, Zur from Synechococcus sp. WH8102 was sub-cloned and purified in the absence of zinc ions in the cultures. Synechococcus sp. WH8102 Zur protein at neutral pH (~pH 7.8), in mass spectrometry, presented as a mixture of species including a monomer with two zinc ions bound Zn2Zur, a dimer with four zinc ions bound, and another well folded Zn4Zur2 dimer. The complementary technique, ICP-OES, confirmed that the zinc binding stoichiometry was in agreement with mass spectrometric findings, 2.1±0.2 Zn(II) ions per monomer. Mimicking a drop in available cellular zinc as may be expected in zinc-deplete conditions, it was found that only one of the two metal ions bound per monomer was removed by EDTA from Synechococcus sp. WH8102 Zur protein, giving 0.9 ± 0.3 Zn(II) atoms per monomer, with shifting in the monomer/dimer equilibrium toward Zn1Zur species. No large difference in the secondary structure between the metallo-species of the SynZur protein was found in CD spectroscopy. Moreover, Synechococcus sp. WH8102 Zur protein was found to behave in a similar manner to previously studied Fur family proteins, where zinc removal from the sensory site is fully reversible and has the ability to re-establish a (2:1) Zn(II):protein ratio in less than 15 min with 1.9 ± 0.4 Zn(II) atoms per monomer. Similarly, Synechococcus sp. WH8102 Zur was found to act as a transcriptional factor in the presence of zinc ions and bind specifically to 23 AT-rich DNA sequence. The “free” cytosolic zinc concentrations that Zur protein trigger transcription of znuABC and smtA found to be in femtomolar range ~ 1.78 ×10-15, with the dissociation constant ~ 62.65±1.47 nM and 61.76 ± 2.42 nM for Zur-PznuA promoter and SynZur-PsmtA promoter, respectively