TY - JOUR T1 - Automation aided optimization of cloning, expression and purification of enzymes of the bacterial sialic acid catabolic and sialylation pathways enzymes for structural studies. JF - Microb Biotechnol Y1 - 2018 A1 - Bairy, Sneha A1 - Gopalan, Lakshmi Narayanan A1 - Setty, Thanuja Gangi A1 - Srinivasachari, Sathya A1 - Manjunath, Lavanyaa A1 - Kumar, Jay Prakash A1 - Guntupalli, Sai R A1 - Bose, Sucharita A1 - Nayak, Vinod A1 - Ghosh, Swagatha A1 - Sathyanarayanan, Nitish A1 - Caing-Carlsson, Rhawnie A1 - Wahlgren, Weixiao Yuan A1 - Friemann, Rosmarie A1 - Ramaswamy, S A1 - Neerathilingam, Muniasamy AB -

The process of obtaining a well-expressing, soluble and correctly folded constructs can be made easier and quicker by automating the optimization of cloning, expression and purification. While there are many semiautomated pipelines available for cloning, expression and purification, there is hardly any pipeline that involves complete automation. Here, we achieve complete automation of all the steps involved in cloning and in vivo expression screening. This is demonstrated using 18 genes involved in sialic acid catabolism and the surface sialylation pathway. Our main objective was to clone these genes into a His-tagged Gateway vector, followed by their small-scale expression optimization in vivo. The constructs that showed best soluble expression were then selected for purification studies and scaled up for crystallization studies. Our technique allowed us to quickly find conditions for producing significant quantities of soluble proteins in Escherichia coli, their large-scale purification and successful crystallization of a number of these proteins. The method can be implemented in other cases where one needs to screen a large number of constructs, clones and expression vectors for successful recombinant production of functional proteins.

VL - 11 IS - 2 ER - TY - JOUR T1 - Crystal structures and kinetics of N-acetylneuraminate lyase from Fusobacterium nucleatum. JF - Acta Crystallogr F Struct Biol Commun Y1 - 2018 A1 - Kumar, Jay Prakash A1 - Rao, Harshvardhan A1 - Nayak, Vinod A1 - Ramaswamy, S KW - Bacterial Proteins KW - Crystallography, X-Ray KW - Fusobacterium nucleatum KW - Hydrogen Bonding KW - Models, Molecular KW - N-Acetylneuraminic Acid KW - Oxo-Acid-Lyases KW - Protein Conformation KW - Protein Folding KW - Pyruvic Acid KW - Schiff Bases KW - Sequence Alignment KW - Tyrosine AB -

N-Acetyl-D-neuraminic acid lyase (NanA) catalyzes the breakdown of sialic acid (Neu5Ac) to N-acetyl-D-mannosamine (ManNAc) and pyruvate. NanA plays a key role in Neu5Ac catabolism in many pathogenic and bacterial commensals where sialic acid is available as a carbon and nitrogen source. Several pathogens or commensals decorate their surfaces with sialic acids as a strategy to escape host innate immunity. Catabolism of sialic acid is key to a range of host-pathogen interactions. In this study, atomic resolution structures of NanA from Fusobacterium nucleatum (FnNanA) in ligand-free and ligand-bound forms are reported at 2.32 and 1.76 Å resolution, respectively. F. nucleatum is a Gram-negative pathogen that causes gingival and periodontal diseases in human hosts. Like other bacterial N-acetylneuraminate lyases, FnNanA also shares the triosephosphate isomerase (TIM)-barrel fold. As observed in other homologous enzymes, FnNanA forms a tetramer. In order to characterize the structure-function relationship, the steady-state kinetic parameters of the enzyme are also reported.

VL - 74 IS - Pt 11 ER - TY - JOUR T1 - Decreased expression of cell adhesion genes in cancer stem-like cells isolated from primary oral squamous cell carcinomas. JF - Tumour Biol Y1 - 2018 A1 - Mishra, Amrendra A1 - Sriram, Harshini A1 - Chandarana, Pinal A1 - Tanavde, Vivek A1 - Kumar, Rekha V A1 - Gopinath, Ashok A1 - Govindarajan, Raman A1 - Ramaswamy, S A1 - Sadasivam, Subhashini AB -

The goal of this study was to isolate cancer stem-like cells marked by high expression of CD44, a putative cancer stem cell marker, from primary oral squamous cell carcinomas and identify distinctive gene expression patterns in these cells. From 1 October 2013 to 4 September 2015, 76 stage III-IV primary oral squamous cell carcinoma of the gingivobuccal sulcus were resected. In all, 13 tumours were analysed by immunohistochemistry to visualise CD44-expressing cells. Expression of CD44 within The Cancer Genome Atlas-Head and Neck Squamous Cell Carcinoma RNA-sequencing data was also assessed. Seventy resected tumours were dissociated into single cells and stained with antibodies to CD44 as well as CD45 and CD31 (together referred as Lineage/Lin). From 45 of these, CD44Lin and CD44Lin subpopulations were successfully isolated using fluorescence-activated cell sorting, and good-quality RNA was obtained from 14 such sorted pairs. Libraries from five pairs were sequenced and the results analysed using bioinformatics tools. Reverse transcription quantitative polymerase chain reaction was performed to experimentally validate the differential expression of selected candidate genes identified from the transcriptome sequencing in the same 5 and an additional 9 tumours. CD44 was expressed on the surface of poorly differentiated tumour cells, and within the The Cancer Genome Atlas-Head and Neck Squamous Cell Carcinoma samples, its messenger RNA levels were higher in tumours compared to normal. Transcriptomics revealed that 102 genes were upregulated and 85 genes were downregulated in CD44Lin compared to CD44Lin cells in at least 3 of the 5 tumours sequenced. The upregulated genes included those involved in immune regulation, while the downregulated genes were enriched for genes involved in cell adhesion. Decreased expression of PCDH18, MGP, SPARCL1 and KRTDAP was confirmed by reverse transcription quantitative polymerase chain reaction. Lower expression of the cell-cell adhesion molecule PCDH18 correlated with poorer overall survival in the The Cancer Genome Atlas-Head and Neck Squamous Cell Carcinoma data highlighting it as a potential negative prognostic factor in this cancer.

VL - 40 IS - 5 ER - TY - JOUR T1 - "Just a spoonful of sugar...": import of sialic acid across bacterial cell membranes. JF - Biophys Rev Y1 - 2018 A1 - North, Rachel A A1 - Horne, Christopher R A1 - Davies, James S A1 - Remus, Daniela M A1 - Muscroft-Taylor, Andrew C A1 - Goyal, Parveen A1 - Wahlgren, Weixiao Yuan A1 - Ramaswamy, S A1 - Friemann, Rosmarie A1 - Dobson, Renwick C J AB -

Eukaryotic cell surfaces are decorated with a complex array of glycoconjugates that are usually capped with sialic acids, a large family of over 50 structurally distinct nine-carbon amino sugars, the most common member of which is N-acetylneuraminic acid. Once made available through the action of neuraminidases, bacterial pathogens and commensals utilise host-derived sialic acid by degrading it for energy or repurposing the sialic acid onto their own cell surface to camouflage the bacterium from the immune system. A functional sialic acid transporter has been shown to be essential for the uptake of sialic acid in a range of human bacterial pathogens and important for host colonisation and persistence. Here, we review the state-of-play in the field with respect to the molecular mechanisms by which these bio-nanomachines transport sialic acids across bacterial cell membranes.

VL - 10 IS - 2 ER - TY - JOUR T1 - Reflections on current Ayurveda research. JF - J Ayurveda Integr Med Y1 - 2018 A1 - Ramaswamy, S AB -

The current development in modern biology partnered with technology, better understanding of genes, environment is beginning to allow predicting the state of the human body. Research in Modern science is in transitional state from reverse pharmacology to system approach. It's time for Ayurveda to undertake research deep in its own foundational theories and in its interface with modern science. The present environment, lifestyle and nutrition have drastically different from ancient times. There is a need to modernize Ayurveda and make it relevant and contextual in terms of personalized medicine where allopathic medicine is heading. Innovations based on advancements, new treatment regimen, therapeutic approaches are the current needs from Ayurveda to make an impact on global clinical practice. In India, the Ayurveda research needs commitment in leadership and good funding resources for its best run, and for true healthcare.

VL - 9 IS - 4 ER - TY - JOUR T1 - The Sodium Sialic Acid Symporter From Has Altered Substrate Specificity. JF - Front Chem Y1 - 2018 A1 - North, Rachel A A1 - Wahlgren, Weixiao Y A1 - Remus, Daniela M A1 - Scalise, Mariafrancesca A1 - Kessans, Sarah A A1 - Dunevall, Elin A1 - Claesson, Elin A1 - Soares da Costa, Tatiana P A1 - Perugini, Matthew A A1 - Ramaswamy, S A1 - Allison, Jane R A1 - Indiveri, Cesare A1 - Friemann, Rosmarie A1 - Dobson, Renwick C J AB -

Mammalian cell surfaces are decorated with complex glycoconjugates that terminate with negatively charged sialic acids. Commensal and pathogenic bacteria can use host-derived sialic acids for a competitive advantage, but require a functional sialic acid transporter to import the sugar into the cell. This work investigates the sodium sialic acid symporter (SiaT) from (SiaT). We demonstrate that SiaT rescues an strain lacking its endogenous sialic acid transporter when grown on the sialic acids -acetylneuraminic acid (Neu5Ac) or -glycolylneuraminic acid (Neu5Gc). We then develop an expression, purification and detergent solubilization system for SiaT and demonstrate that the protein is largely monodisperse in solution with a stable monomeric oligomeric state. Binding studies reveal that SiaT has a higher affinity for Neu5Gc over Neu5Ac, which was unexpected and is not seen in another SiaT homolog. We develop a homology model and use comparative sequence analyses to identify substitutions in the substrate-binding site of SiaT that may explain the altered specificity. SiaT is shown to be electrogenic, and transport is dependent upon more than one Na ion for every sialic acid molecule. A functional sialic acid transporter is essential for the uptake and utilization of sialic acid in a range of pathogenic bacteria, and developing new inhibitors that target these transporters is a valid mechanism for inhibiting bacterial growth. By demonstrating a route to functional recombinant SiaT, and developing the and assay systems, our work underpins the design of inhibitors to this transporter.

VL - 6 ER - TY - JOUR T1 - A strategy to identify a ketoreductase that preferentially synthesizes pharmaceutically relevant (S)-alcohols using whole-cell biotransformation. JF - Microb Cell Fact Y1 - 2018 A1 - Haq, Saiful F A1 - Shanbhag, Anirudh P A1 - Karthikeyan, Subbulakshmi A1 - Hassan, Imran A1 - Thanukrishnan, Kannan A1 - Ashok, Abhishek A1 - Sukumaran, Sunilkumar A1 - Ramaswamy, S A1 - Bharatham, Nagakumar A1 - Datta, Santanu A1 - Samant, Shalaka A1 - Katagihallimath, Nainesh KW - Biotransformation KW - Catalysis KW - Ethanol KW - Ketones AB -

INTRODUCTION: Chemical industries are constantly in search of an expeditious and environmentally benign method for producing chiral synthons. Ketoreductases have been used as catalysts for enantioselective conversion of desired prochiral ketones to their corresponding alcohol. We chose reported promiscuous ketoreductases belonging to different protein families and expressed them in E. coli to evaluate their ability as whole-cell catalysts for obtaining chiral alcohol intermediates of pharmaceutical importance. Apart from establishing a method to produce high value (S)-specific alcohols that have not been evaluated before, we propose an in silico analysis procedure to predict product chirality.

RESULTS: Six enzymes originating from Sulfolobus sulfotaricus, Zygosaccharomyces rouxii, Hansenula polymorpha, Corynebacterium sp. ST-10, Synechococcus sp. PCC 7942 and Bacillus sp. ECU0013 with reported efficient activity for dissimilar substrates are compared here to arrive at an optimal enzyme for the method. Whole-cell catalysis of ketone intermediates for drugs like Aprepitant, Sitagliptin and Dolastatin using E. coli over-expressing these enzymes yielded (S)-specific chiral alcohols. We explain this chiral specificity for the best-performing enzyme, i.e., Z. rouxii ketoreductase using in silico modelling and MD simulations. This rationale was applied to five additional ketones that are used in the synthesis of Crizotinib, MA-20565 (an antifungal agent), Sulopenem, Rivastigmine, Talampanel and Barnidipine and predicted the yield of (S) enantiomers. Experimental evaluation matched the in silico analysis wherein ~ 95% (S)-specific alcohol with a chemical yield of 23-79% was obtained through biotransformation. Further, the cofactor re-cycling was optimized by switching the carbon source from glucose to sorbitol that improved the chemical yield to 85-99%.

CONCLUSIONS: Here, we present a strategy to synthesize pharmaceutically relevant chiral alcohols by ketoreductases using a cofactor balanced whole-cell catalysis scheme that is useful for the industry. Based on the results obtained in these trials, Zygosaccharomyces rouxii ketoreductase was identified as a proficient enzyme to obtain (S)-specific alcohols from their respective ketones. The whole-cell catalyst when combined with nutrient modulation of using sorbitol as a carbon source helped obtain high enantiomeric and chemical yield.

VL - 17 IS - 1 ER - TY - JOUR T1 - Substrate-bound outward-open structure of a Na-coupled sialic acid symporter reveals a new Na site. JF - Nat Commun Y1 - 2018 A1 - Wahlgren, Weixiao Y A1 - Dunevall, Elin A1 - North, Rachel A A1 - Paz, Aviv A1 - Scalise, Mariafrancesca A1 - Bisignano, Paola A1 - Bengtsson-Palme, Johan A1 - Goyal, Parveen A1 - Claesson, Elin A1 - Caing-Carlsson, Rhawnie A1 - Andersson, Rebecka A1 - Beis, Konstantinos A1 - Nilsson, Ulf J A1 - Farewell, Anne A1 - Pochini, Lorena A1 - Indiveri, Cesare A1 - Grabe, Michael A1 - Dobson, Renwick C J A1 - Abramson, Jeff A1 - Ramaswamy, S A1 - Friemann, Rosmarie AB -

Many pathogenic bacteria utilise sialic acids as an energy source or use them as an external coating to evade immune detection. As such, bacteria that colonise sialylated environments deploy specific transporters to mediate import of scavenged sialic acids. Here, we report a substrate-bound 1.95 Å resolution structure and subsequent characterisation of SiaT, a sialic acid transporter from Proteus mirabilis. SiaT is a secondary active transporter of the sodium solute symporter (SSS) family, which use Na gradients to drive the uptake of extracellular substrates. SiaT adopts the LeuT-fold and is in an outward-open conformation in complex with the sialic acid N-acetylneuraminic acid and two Na ions. One Na binds to the conserved Na2 site, while the second Na binds to a new position, termed Na3, which is conserved in many SSS family members. Functional and molecular dynamics studies validate the substrate-binding site and demonstrate that both Na sites regulate N-acetylneuraminic acid transport.

VL - 9 IS - 1 ER - TY - JOUR T1 - Crystal structure of N-acetylmannosamine kinase from Fusobacterium nucleatum. JF - Acta Crystallogr F Struct Biol Commun Y1 - 2017 A1 - Caing-Carlsson, Rhawnie A1 - Goyal, Parveen A1 - Sharma, Amit A1 - Ghosh, Swagatha A1 - Setty, Thanuja Gangi A1 - North, Rachel A A1 - Friemann, Rosmarie A1 - Ramaswamy, S KW - Adenosine Triphosphate KW - Amino Acid Sequence KW - Bacterial Proteins KW - Binding Sites KW - Cloning, Molecular KW - Crystallography, X-Ray KW - Escherichia coli KW - Fusobacterium nucleatum KW - Gene Expression KW - Genetic Vectors KW - Hexosamines KW - Models, Molecular KW - Phosphotransferases (Alcohol Group Acceptor) KW - Protein Binding KW - Protein Conformation, alpha-Helical KW - Protein Conformation, beta-Strand KW - Protein Interaction Domains and Motifs KW - Protein Multimerization KW - Recombinant Proteins KW - Sequence Alignment KW - Sequence Homology, Amino Acid KW - Substrate Specificity AB -

Sialic acids comprise a varied group of nine-carbon amino sugars that are widely distributed among mammals and higher metazoans. Some human commensals and bacterial pathogens can scavenge sialic acids from their environment and degrade them for use as a carbon and nitrogen source. The enzyme N-acetylmannosamine kinase (NanK; EC 2.7.1.60) belongs to the transcriptional repressors, uncharacterized open reading frames and sugar kinases (ROK) superfamily. NanK catalyzes the second step of the sialic acid catabolic pathway, transferring a phosphate group from adenosine 5'-triphosphate to the C6 position of N-acetylmannosamine to generate N-acetylmannosamine 6-phosphate. The structure of NanK from Fusobacterium nucleatum was determined to 2.23 Å resolution by X-ray crystallography. Unlike other NanK enzymes and ROK family members, F. nucleatum NanK does not have a conserved zinc-binding site. In spite of the absence of the zinc-binding site, all of the major structural features of enzymatic activity are conserved.

VL - 73 IS - Pt 6 ER - TY - JOUR T1 - Horse Liver Alcohol Dehydrogenase: Zinc Coordination and Catalysis. JF - Biochemistry Y1 - 2017 A1 - Plapp, Bryce V A1 - Savarimuthu, Baskar Raj A1 - Ferraro, Daniel J A1 - Rubach, Jon K A1 - Brown, Eric N A1 - Ramaswamy, S KW - 2,2'-Dipyridyl KW - Adenosine Diphosphate Ribose KW - Alcohol Dehydrogenase KW - Animals KW - Catalytic Domain KW - Crystallography, X-Ray KW - Formamides KW - Horses KW - Kinetics KW - Liver KW - Models, Molecular KW - NAD KW - Phenanthrolines KW - Protein Binding KW - Protein Conformation KW - Water KW - Zinc AB -

During catalysis by liver alcohol dehydrogenase (ADH), a water bound to the catalytic zinc is replaced by the oxygen of the substrates. The mechanism might involve a pentacoordinated zinc or a double-displacement reaction with participation by a nearby glutamate residue, as suggested by studies of human ADH3, yeast ADH1, and some other tetrameric ADHs. Zinc coordination and participation of water in the enzyme mechanism were investigated by X-ray crystallography. The apoenzyme and its complex with adenosine 5'-diphosphoribose have an open protein conformation with the catalytic zinc in one position, tetracoordinated by Cys-46, His-67, Cys-174, and a water molecule. The bidentate chelators 2,2'-bipyridine and 1,10-phenanthroline displace the water and form a pentacoordinated zinc. The enzyme-NADH complex has a closed conformation similar to that of ternary complexes with coenzyme and substrate analogues; the coordination of the catalytic zinc is similar to that found in the apoenzyme, except that a minor, alternative position for the catalytic zinc is ∼1.3 Å from the major position and closer to Glu-68, which could form the alternative coordination to the catalytic zinc. Complexes with NADH and N-1-methylhexylformamide or N-benzylformamide (or with NAD and fluoro alcohols) have the classical tetracoordinated zinc, and no water is bound to the zinc or the nicotinamide rings. The major forms of the enzyme in the mechanism have a tetracoordinated zinc, where the carboxylate group of Glu-68 could participate in the exchange of water and substrates on the zinc. Hydride transfer in the Michaelis complexes does not involve a nearby water.

VL - 56 IS - 28 ER - TY - JOUR T1 - Structural and functional studies of ferredoxin and oxygenase components of 3-nitrotoluene dioxygenase from Diaphorobacter sp. strain DS2. JF - PLoS One Y1 - 2017 A1 - Kumari, Archana A1 - Singh, Deepak A1 - Ramaswamy, S A1 - Ramanathan, Gurunath KW - Catalytic Domain KW - Comamonadaceae KW - Crystallography, X-Ray KW - Ferredoxins KW - Molecular Docking Simulation KW - Mutation KW - Oxygenases KW - Substrate Specificity KW - Toluene AB -

3-nitrotoluene dioxygenase (3NTDO) from Diaphorobacter sp. strain DS2 catalyses the conversion of 3-nitrotoluene (3NT) into a mixture of 3- and 4-methylcatechols with release of nitrite. We report here, X-ray crystal structures of oxygenase and ferredoxin components of 3NTDO at 2.9 Å and 2.4 Å, respectively. The residues responsible for nitrite release in 3NTDO were further probed by four single and two double mutations in the catalytic site of α-subunit of the dioxygenase. Modification of Val 350 to Phe, Ile 204 to Ala, and Asn258 to Val by site directed mutagenesis resulted in inactive enzymes revealing the importance of these residues in catalysis. Docking studies of meta nitrotoluene to the active site of 3NTDO suggested possible orientations of binding that favor the formation of 3-methylcatechol (3MC) over 4-methylcatechol energetically. The electron transfer pathway from ferredoxin subunit to the active site of the oxygenase subunit is also proposed.

VL - 12 IS - 4 ER - TY - JOUR T1 - Blue protein with red fluorescence. JF - Proc Natl Acad Sci U S A Y1 - 2016 A1 - Ghosh, Swagatha A1 - Yu, Chi-Li A1 - Ferraro, Daniel J A1 - Sudha, Sai A1 - Pal, Samir Kumar A1 - Schaefer, Wayne F A1 - Gibson, David T A1 - Ramaswamy, S KW - Biliverdine KW - Crystallography, X-Ray KW - Fluorescence KW - Models, Molecular KW - Proteins KW - Recombinant Proteins AB -

The walleye (Sander vitreus) is a golden yellow fish that inhabits the Northern American lakes. The recent sightings of the blue walleye and the correlation of its sighting to possible increased UV radiation have been proposed earlier. The underlying molecular basis of its adaptation to increased UV radiation is the presence of a protein (Sandercyanin)-ligand complex in the mucus of walleyes. Degradation of heme by UV radiation results in the formation of Biliverdin IXα (BLA), the chromophore bound to Sandercyanin. We show that Sandercyanin is a monomeric protein that forms stable homotetramers on addition of BLA to the protein. A structure of the Sandercyanin-BLA complex, purified from the fish mucus, reveals a glycosylated protein with a lipocalin fold. This protein-ligand complex absorbs light in the UV region (λ of 375 nm) and upon excitation at this wavelength emits in the red region (λ of 675 nm). Unlike all other known biliverdin-bound fluorescent proteins, the chromophore is noncovalently bound to the protein. We provide here a molecular rationale for the observed spectral properties of Sandercyanin.

VL - 113 IS - 41 ER - TY - JOUR T1 - Mechanistic implications from structures of yeast alcohol dehydrogenase complexed with coenzyme and an alcohol. JF - Arch Biochem Biophys Y1 - 2016 A1 - Plapp, Bryce V A1 - Charlier, Henry A A1 - Ramaswamy, S KW - Alcohol Dehydrogenase KW - Binding Sites KW - Catalysis KW - Coenzymes KW - Computer Simulation KW - Enzyme Activation KW - Models, Chemical KW - Models, Molecular KW - NAD KW - Protein Binding KW - Protein Conformation KW - Saccharomyces cerevisiae Proteins KW - Substrate Specificity KW - Trifluoroethanol AB -

Yeast alcohol dehydrogenase I is a homotetramer of subunits with 347 amino acid residues, catalyzing the oxidation of alcohols using NAD(+) as coenzyme. A new X-ray structure was determined at 3.0 Å where both subunits of an asymmetric dimer bind coenzyme and trifluoroethanol. The tetramer is a pair of back-to-back dimers. Subunit A has a closed conformation and can represent a Michaelis complex with an appropriate geometry for hydride transfer between coenzyme and alcohol, with the oxygen of 2,2,2-trifluoroethanol ligated at 2.1 Å to the catalytic zinc in the classical tetrahedral coordination with Cys-43, Cys-153, and His-66. Subunit B has an open conformation, and the coenzyme interacts with amino acid residues from the coenzyme binding domain, but not with residues from the catalytic domain. Coenzyme appears to bind to and dissociate from the open conformation. The catalytic zinc in subunit B has an alternative, inverted coordination with Cys-43, Cys-153, His-66 and the carboxylate of Glu-67, while the oxygen of trifluoroethanol is 3.5 Å from the zinc. Subunit B may represent an intermediate in the mechanism after coenzyme and alcohol bind and before the conformation changes to the closed form and the alcohol oxygen binds to the zinc and displaces Glu-67.

VL - 591 ER -