@article {122349, title = {A dimer between monomers and hexamers-Oligomeric variations in glucosamine-6-phosphate deaminase family.}, journal = {PLoS One}, volume = {18}, year = {2023}, month = {2023}, pages = {e0271654}, abstract = {

In bacteria that live in hosts whose terminal sugar is a sialic acid, Glucosamine-6-phosphate deaminase (NagB) catalyzes the last step in converting sialic acid into Fructose-6-phosphate. These bacteria then use the Fructose-6-phosphate as an energy source. The enzyme NagB exists as a hexamer in Gram-negative bacteria and is allosterically regulated. In Gram-positive bacteria, it exists as a monomer and lacks allosteric regulation. Our identification of a dimeric Gram-negative bacterial NagB motivated us to characterize the structural basis of two closely related oligomeric forms. We report here the crystal structures of NagB from two Gram-negative pathogens, Haemophilus influenzae (Hi) and Pasturella multocida (Pm). The Hi-NagB is active as a hexamer, while Pm-NagB is active as a dimer. Both Hi-NagB and Pm-NagB contain the C-terminal helix implicated as essential for hexamer formation. The hexamer is described as a dimer of trimers. In the Pm-NagB dimer, the dimeric interface is conserved. The conservation of the dimer interface suggests that the three possible oligomeric forms of NagB are a monomer, a dimer, and a trimer of dimers. Computational modeling and MD simulations indicate that the residues at the trimeric interface have less stabilizing energy of oligomer formation than those in the dimer interface. We propose that Pm-NagB is the evolutionary link between the monomer and the hexamer forms.

}, keywords = {Aldose-Ketose Isomerases, Bacterial Proteins, Haemophilus influenzae, N-Acetylneuraminic Acid, Pasteurella multocida, Polymers}, issn = {1932-6203}, doi = {10.1371/journal.pone.0271654}, author = {Srinivasachari, Sathya and Tiwari, Vikas R and Kharbanda, Tripti and Sowdamini, Ramanathan and Subramanian, Ramaswamy} } @article {79721, title = {A 2-Tyr-1-carboxylate Mononuclear Iron Center Forms the Active Site of a Paracoccus Dimethylformamidase.}, journal = {Angew Chem Int Ed Engl}, year = {2020}, month = {2020 May 25}, abstract = {

N,N-dimethyl formamide (DMF) is an extensively used organic solvent but is also a potent pollutant. Certain bacterial species from genera such as Paracoccus, Pseudomonas, and Alcaligenes have evolved to use DMF as a sole carbon and nitrogen source for growth via degradation by a dimethylformamidase (DMFase). We show that DMFase from Paracoccus sp. strain DMF is a halophilic and thermostable enzyme comprising a multimeric complex of the α β or (α β ) type. One of the three domains of the large subunit and the small subunit are hitherto undescribed protein folds of unknown evolutionary origin. The active site consists of a mononuclear iron coordinated by two Tyr side-chain phenolates and one carboxylate from Glu. The Fe ion in the active site catalyzes the hydrolytic cleavage of the amide bond in DMF. Kinetic characterization reveals that the enzyme shows cooperativity between subunits, and mutagenesis and structural data provide clues to the catalytic mechanism.

}, issn = {1521-3773}, doi = {10.1002/anie.202005332}, author = {Arya, Chetan Kumar and Yadav, Swati and Fine, Jonathan and Casanal, Ana and Chopra, Gaurav and Ramanathan, Gurunath and Vinothkumar, Kutti R and Subramanian, Ramaswamy} } @article {80091, title = {Comparison of CryoEM and X-ray structures of dimethylformamidase.}, journal = {Prog Biophys Mol Biol}, year = {2020}, month = {2020 Jul 28}, abstract = {

Dimethylformamidase (DMFase) catalyzes the hydrolysis of dimethylformamide, an industrial solvent, introduced into the environment by humans. Recently, we determined the structures of dimethylformamidase by electron cryo microscopy and X-ray crystallography revealing a tetrameric enzyme with a mononuclear iron at the active site. DMFase from Paracoccus sp. isolated from a waste water treatment plant around the city of Kanpur in India shows maximal activity at 54 {\textdegree}C and is halotolerant. The structures determined by both techniques are mostly identical and the largest difference is in a loop near the active site. This loop could play a role in co-operativity between the monomers. A number of non-protein densities are observed in the EM map, which are modelled as water molecules. Comparison of the structures determined by the two methods reveals conserved water molecules that could play a structural role. The higher stability, unusual active site and negligible activity at low temperature makes this a very good model to study enzyme mechanism by cryoEM.

}, issn = {1873-1732}, doi = {10.1016/j.pbiomolbio.2020.06.008}, author = {Vinothkumar, Kutti R and Arya, Chetan and Ramanathan, Gurunath and Subramanian, Ramaswamy} } @article {86518, title = {Structure and Function of -Acetylmannosamine Kinases from Pathogenic Bacteria.}, journal = {ACS Omega}, volume = {5}, year = {2020}, month = {2020 Dec 08}, pages = {30923-30936}, abstract = {

Several pathogenic bacteria import and catabolize sialic acids as a source of carbon and nitrogen. Within the sialic acid catabolic pathway, the enzyme -acetylmannosamine kinase (NanK) catalyzes the phosphorylation of -acetylmannosamine to -acetylmannosamine-6-phosphate. This kinase belongs to the ROK superfamily of enzymes, which generally contain a conserved zinc-finger (ZnF) motif that is important for their structure and function. Previous structural studies have shown that the ZnF motif is absent in NanK of (-NanK), a Gram-negative bacterium that causes the gum disease gingivitis. However, the effect in loss of the ZnF motif on the kinase activity is unknown. Using kinetic and thermodynamic studies, we have studied the functional properties of -NanK to its substrates ManNAc and ATP, compared its activity with other ZnF motif-containing NanK enzymes from closely related Gram-negative pathogenic bacteria (-NanK), (-NanK), and (-NanK). Our studies show a 10-fold decrease in substrate binding affinity between NanK (apparent ≈ 700 μM) and ZnF motif-containing NanKs (apparent ≈ 60 μM). To understand the structural features that combat the loss of the ZnF motif in -NanK, we solved the crystal structures of functionally homologous ZnF motif-containing NanKs from and . Here, we report -NanK:unliganded, -NanK:AMPPNP, -NanK:ManNAc, -NanK:ManNAc, and -NanK:ManNAc-6P:ADP crystal structures. Structural comparisons of -NanK with -NanK, -NanK, and hMNK (human -acetylmannosamine kinase domain of UDP--acetylglucosamine-2-epimerase/-acetylmannosamine kinase, GNE) show that even though there is less sequence identity, they have high degree of structural similarity. Furthermore, our structural analyses highlight that the ZnF motif of -NanK is substituted by a set of hydrophobic residues, which forms a hydrophobic cluster that helps the proper orientation of ManNac in the active site. In summary, ZnF-containing and ZnF-lacking NanK enzymes from different Gram-negative pathogenic bacteria are functionally very similar but differ in their metal requirement. Our structural studies unveil the structural modifications in -NanK that compensate the loss of the ZnF motif in comparison to other NanK enzymes.

}, issn = {2470-1343}, doi = {10.1021/acsomega.0c03699}, author = {Gangi Setty, Thanuja and Sarkar, Arunabha and Coombes, David and Dobson, Renwick C J and Subramanian, Ramaswamy} }