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ProX from marine Synechococcus spp. show a sole preference for glycine-betaine with differential affinity between ecotypes.

ABSTRACT: Osmotic stress, caused by high or fluctuating salt concentrations, is a crucial abiotic factor affecting microbial growth in aquatic habitats. Many organisms utilize common responses to osmotic stress, generally requiring active extrusion of toxic inorganic ions and accumulation of compatible solutes to protect cellular machinery. We heterologously expressed and purified predicted osmoprotectant, proline/glycine betaine-binding proteins (ProX) from two phylogenetically distinct Synechococcus spp. MITS9220 and WH8102. Homologues of this protein are conserved only among Prochlorococcus LLIV and Synechococcus clade I, III and CRD1 strains. Our biophysical characterization show Synechococcus ProX exists as a dimer, with specificity solely for glycine betaine but not to other osmoprotectants tested. We discovered that MITS9220_ProX has a 10-fold higher affinity to glycine betaine than WH8102_ProX, which is further elevated (24-fold) in high salt conditions. The stronger affinity and effect of ionic strength on MITS9220_ProX glycine betaine binding but not on WH8102_ProX alludes to a novel regulatory mechanism, providing critical functional insights into the phylogenetic divergence of picocyanobacterial ProX proteins that may be necessary for their ecological success.

SUBMITTER: Ford BA

PROVIDER: S-EPMC10087775 | biostudies-literature | 2022 Dec

REPOSITORIES: biostudies-literature

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ProX from marine Synechococcus spp. show a sole preference for glycine-betaine with differential affinity between ecotypes.

Ford Benjamin A BA Ranjit Pramita P Mabbutt Bridget C BC Paulsen Ian T IT Shah Bhumika S BS

Environmental microbiology 20220828 12

Osmotic stress, caused by high or fluctuating salt concentrations, is a crucial abiotic factor affecting microbial growth in aquatic habitats. Many organisms utilize common responses to osmotic stress, generally requiring active extrusion of toxic inorganic ions and accumulation of compatible solutes to protect cellular machinery. We heterologously expressed and purified predicted osmoprotectant, proline/glycine betaine-binding proteins (ProX) from two phylogenetically distinct Synechococcus spp ...[more]

PMID: 36054310

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Project description:Although some bacteria, including Chromohalobacter salexigens DSM 3043, can use glycine betaine (GB) as a sole source of carbon and energy, little information is available about the genes and their encoded proteins involved in the initial step of the GB degradation pathway. In the present study, the results of conserved domain analysis, construction of in-frame deletion mutants, and an in vivo functional complementation assay suggested that the open reading frames Csal_1004 and Csal_1005, designated bmoA and bmoB, respectively, may act as the terminal oxygenase and the ferredoxin reductase genes in a novel Rieske-type oxygenase system to convert GB to dimethylglycine in C. salexigens DSM 3043. To further verify their function, BmoA and BmoB were heterologously overexpressed in Escherichia coli, and 13C nuclear magnetic resonance analysis revealed that dimethylglycine was accumulated in E. coli BL21(DE3) expressing BmoAB or BmoA. In addition, His-tagged BmoA and BmoB were individually purified to electrophoretic homogeneity and estimated to be a homotrimer and a monomer, respectively. In vitro biochemical analysis indicated that BmoB is an NADH-dependent flavin reductase with one noncovalently bound flavin adenine dinucleotide (FAD) as its prosthetic group. In the presence of BmoB, NADH, and flavin, BmoA could aerobically degrade GB to dimethylglycine with the concomitant production of formaldehyde. BmoA exhibited strict substrate specificity for GB, and its demethylation activity was stimulated by Fe2+ Phylogenetic analysis showed that BmoA belongs to group V of the Rieske nonheme iron oxygenase (RO) family, and all the members in this group were able to use quaternary ammonium compounds as substrates.IMPORTANCE GB is widely distributed in nature. In addition to being accumulated intracellularly as a compatible solute to deal with osmotic stress, it can be utilized by many bacteria as a source of carbon and energy. However, very limited knowledge is presently available about the molecular and biochemical mechanisms for the initial step of the aerobic GB degradation pathway in bacteria. Here, we report the molecular and biochemical characterization of a novel two-component Rieske-type monooxygenase system, GB monooxygenase (BMO), which is responsible for oxidative demethylation of GB to dimethylglycine in C. salexigens DSM 3043. The results gained in this study extend our knowledge on the catalytic reaction of microbial GB degradation to dimethylglycine.

Project description:Folate, or vitamin B9, is an important compound in one-carbon metabolism. Previous studies have found weaker binding of dihydrofolate to dihydrofolate reductase in the presence of osmolytes. In other words, osmolytes are more difficult to remove from the dihydrofolate solvation shell than water; this shifts the equilibrium toward the free ligand and protein species. This study uses vapor-pressure osmometry to explore the interaction of folate with the model osmolyte, glycine betaine. This method yields a preferential interaction potential (μ23/RT value). This value is concentration-dependent as folate dimerizes. The μ23/RT value also tracks the deprotonation of folate's N3-O4 keto-enol group, yielding a pKa of 8.1. To determine which folate atoms interact most strongly with betaine, the interaction of heterocyclic aromatic compounds (as well as other small molecules) with betaine was monitored. Using an accessible surface area approach coupled with osmometry measurements, deconvolution of the μ23/RT values into α values for atom types was achieved. This allows prediction of μ23/RT values for larger molecules such as folate. Molecular dynamics simulations of folate show a variety of structures from extended to L-shaped. These conformers possess μ23/RT values from -0.18 to 0.09 m-1, where a negative value indicates a preference for solvation by betaine and a positive value indicates a preference for water. This range of values is consistent with values observed in osmometry and solubility experiments. As the average predicted folate μ23/RT value is near zero, this indicates folate interacts almost equally well with betaine and water. Specifically, the glutamate tail prefers to interact with water, while the aromatic rings prefer betaine. In general, the more protonated species in our small molecule survey interact better with betaine as they provide a source of hydrogens (betaine is not a hydrogen bond donor). Upon deprotonation of the small molecule, the preference swings toward water interaction because of its hydrogen bond donating capacities.

Dataset Information

ProX from marine Synechococcus spp. show a sole preference for glycine-betaine with differential affinity between ecotypes.

Publications

ProX from marine Synechococcus spp. show a sole preference for glycine-betaine with differential affinity between ecotypes.

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