Mechanistic Insights Into The Recognition And Catalysis Of Several Important Oceanic Organic Matters By Bacterial Functional Proteins

Microorganisms play important roles in marine nitrogen and sulfur cycles. Peptide uptake plays a significant role in nutrition supply for marine bacteria. It is also an important step in marine nitrogen cycling. Trimethylamine N-oxide （TMAO） and trimethylamine （TMA） are important nitrogen source for marine bacteria. Bacterial TMA monooxygenase can oxidize TMA to TMAO. Dimethylsulfoniopropionate （DMSP） is produced worldwide in a large amount,～109 tons per annum. Dimethyl sulfide （DMS） is produced mainly through the cleavage of DMSP by DMSP lyases. DMS is an important participant in the global sulfur cycle, and DMS oxidation products may influence weather and climate. In this study, we focused on the underlying molecular mechanisms of some key process involved in marine nitrogen and sulfur cycles, including the recognition mechanism of dipeptide by marine bacteria, the recognition mechanism of TMAO by marine bacteria, the molecular mechanism of bacterial oxidation of oceanic TMA into TMAO, and the molecular mechanism of bacterial cleavage of oceanic DMSP into DMS.1. Mutispecific recognition mechanism of dipeptide by marine bacteriaPeptide uptake is important for nutrition supply for marine bacteria. It is also an important step in marine nitrogen cycling. However, how marine bacteria absorb peptides is still not fully understood. DppA is the periplasmic dipeptide binding protein of dipeptide permease （Dpp, an important peptide transporter in bacteria）, and exclusively controls the substrate specificity of Dpp. Here, the substrate binding specificity of deep-sea Pseudoalteromonas sp. SM9913 DppA （PsDppA） was analyzed for 25 different dipeptides with varying properties by using isothermal titration calorimetry measurements. PsDppA showed binding affinities for 8 dipeptides. To explain the multispecific substrate recognition mechanism of PsDppA, we solved the crystal structures of unliganded PsDppA and of PsDppA in complex with 4 different types of dipeptides （Ala-Phe, Met-Leu, Gly-Glu and Val-Thr）. PsDppA alternates between an "open" and a "closed" form during substrate binding. Structural analyses of the 4 PsDppA-substrate complexes combined with mutational assays indicate that PsDppA binds to different substrates through a precise mechanism:dipeptides are bound mainly by the interactions between their backbones and PsDppA, in particular by anchoring their N- and C- termini through ion-pair interactions; hydrophobic interactions are important in binding hydrophobic dipeptides; Lys457 is necessary for the binding of dipeptides with a C-terminal glutamic acid or glutamine. Additionally, sequence alignment suggests that the substrate recognition mechanism of PsDppA may be common in Gram-negative bacteria. Altogether, our results provide structural insights into the multispecific substrate recognition mechanism of marine Gram-negative bacterial DppA, which provides a better understanding of the mechanisms of marine bacterial peptide uptake.2. Recognition mechanism of TMAO by marine bacteriaTMAO is an important nitrogen source for marine bacteria. TMAO can also be metabolized by marine bacteria into volatile methylated amines, the precursors of the greenhouse gas nitrous oxide. However, it was not known how TMAO is recognized and imported by bacteria. Ruegeria pomeroyi DSS-3, a marine Roseobacter, has an ATP-binding cassette （ABC） transporter, TmoXWV, specific for TMAO. TmoX is the substrate-binding protein of the TmoXWV transporter. In this study, the substrate specificity of TmoX of R. pomeroyi DSS-3 was characterized. The recombinate TmoX exhibited a high binding affinity to TMAO in vitro. We further determined the structure of the TmoX/TMAO complex and studied the TMAO-binding mechanism of TmoX by biochemical, structural and mutational analyses. A Ca2+ chelated by an extended loop in TmoX was shown to be important for maintaining the stability of TmoX. Molecular dynamics simulations indicate that TmoX can alternate between "open" and "closed" states for binding TMAO. In the substrate-binding pocket, four tryptophan residues interact with the quaternary amine of TMAO by cation-π interactions, and Glu131 forms a hydrogen bond with the polar oxygen atom of TMAO. The π-π stacking interactions between the side chains of Phe and Tip are also essential for TMAO binding. Sequence analysis suggests that the TMAO-binding mechanism of TmoX may have universal significance in marine bacteria, especially in the marine Roseobacter clade. This study sheds light on how marine microorganisms utilize TMAO.3. Molecular mechanism of bacterial oxidation of oceanic TMA into TMAOTMA and TMAO are widespread in the ocean, and represent important nitrogen source for marine bacteria. Bacterial flavin-containing monooxygenase （FMO）, termed Tmm, can oxidize TMA to TMAO. The corresponding gene is abundant in marine bacteria. Tmm plays an important role in marine carbon and nitrogen cycles. However, the molecular mechanism of the oxidation of TMA by Tmm is still unclear. In this study, Tmm from Roseovarius nubinhibens ISM was characterized. The optimal temperature for Tmm enzymatic activity was ～30℃, and the optimal pH was-8.0. We further determined the crystal structures of Tmm/FAD/NADPH complex, the mutant Tyr207Ser （with very low activity） in complex with FAD and NADPH, and Tyr207Ser /FAD/NADPH complex soaked with TMA. The catalytic process of Tmm can be divided into a reductive half-reaction and an oxidative half-reaction. Structural analysis indicates that the NADP+ molecule generates a conformational change and forms a new hydrogen bond with Tmm residue Asp317 during the oxidative half-reaction, which results in two consequences:Firstly, shutting off the entrance and promoting a protected micro-environment; Secondly, exposing the activated catalytic site of FAD intermediate to TMA to complete the reaction. This is the first structural evidence that NADP+ bounds to Tmm throughout catalysis. Based on our results, a relatively complete catalytic cycle of Tmm to catalyze TMA is proposed. Sequence analysis suggests that the proposed catalytic mechanism of Tmm should be common in bacteria, especially in marine Roseobacter clade and the SAR11 clade. This study provides novel insights into the catalysis of FMOs, and should lead to a better understanding of marine carbon and nitrogen cycling.4. Molecular mechanism of bacterial cleavage of oceanic DMSP into DMSThe microbial cleavage of DMSP generates volatile DMS through the action of DMSP lyases and is important in the global sulfur and carbon cycles. When released into the atmosphere from the oceans, DMS is oxidized, forming cloud condensation nuclei that may influence weather and climate. Six different DMSP lyase genes are found in taxonomically diverse microorganisms, and dddQ is among the most abundant in marine metagenomes. Here, we examine the molecular mechanism of DMSP cleavage by the DMSP lyase, DddQ, from Ruegeria lacuscaerulensis ITI₁57. The enzymatic properties of DddQ was characterized. The optimal temperature for Tmm enzymatic activity was 30℃, and the optimal pH was 8.0. Mn2+ and Co2+ dramatically activated the enzymatic activity of DddQ, whereas Zn2+ almost completely abolished its activity. The structures of DddQ bound to an inhibitory molecule 2-（N-morpholino）ethanesulfonic acid and of DddQ inactivated by a Tyr131Ala mutation and bound to DMSP were solved. DddQ adopts a β-barrel fold structure, and contains a Zn2+ ion and six highly conserved hydrophilic residues （Tyr120, Hisl23, Hisl25, Glu129, Tyr131 and His163） in the active site. Mutational and biochemical analyses indicate that these hydrophilic residues are essential to catalysis. In particular, Tyr131 undergoes a conformational change during catalysis, acting as a base to initiate the β-elimination reaction in DMSP lysis. Moreover, structural analyses and molecular dynamics simulations indicate that two loops over the substrate-binding pocket of DddQ can alternate between "open" and "closed" states, serving as a gate for DMSP entry. We also propose the first molecular mechanism for DMS production through DMSP cleavage. Our study provides important insight into the mechanism involved in the conversion of DMSP into DMS, which should lead to a better understanding of this globally-important biogeochemical reaction.In conclusion, this study revealed the molecular mechanisms of some important physiological processes involved in marine nitrogen and sulfur cycles, which provides a better understanding of marine nitrogen and sulfur cycles.