title: Transglycosylation by chitinase D from Serratia proteamaculans improved through altered substrate interactions.
abstract: We describe the improvement of transglycosylation (TG) by chitinase D from Serratia proteamaculans (SpChiD). The SpChiD produced a smaller quantity of TG products for up to 90 min with 2 mm chitotetraose as the substrate and subsequently produced only hydrolytic products. Of the five residues targeted at the catalytic center, E159D resulted in substantial loss of both hydrolytic and TG activities. Y160A resulted in a product profile similar to SpChiD and a rapid turnover of substrate with slightly increased TG activity. The rest of the three mutants, M226A, Y228A, and R284A, displayed improved TG and decreased hydrolytic ability. Four of the five amino acid substitutions, F64W, F125A, G119S, and S116G, at the catalytic groove increased TG activity, whereas W120A completely lost the TG activity with a concomitant increase in hydrolysis. Mutation of Trp-247 at the solvent-accessible region significantly reduced the hydrolytic activity with increased TG activity. The mutants M226A, Y228A, F125A, S116G, F64W, G119S, R284A, and W247A accumulated approximately double the concentration of TG products like chitopentaose and chitohexaose, compared with SpChiD. The double mutant E159D/F64W regained the activity with accumulation of 6.0% chitopentaose at 6 h, similar to SpChiD at 30 min. Loss of chitobiase activity was unique to Y228A. Substitution of amino acids at the catalytic center and/or groove substantially improved the TG activity of SpChiD, both in terms of the quantity of TG products produced and the extended duration of TG activity.
authors.affiliation: Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Gachibowli, Hyderabad-500046, A.P., India.
authors.forename: Jogi
authors.initials: J
authors.lastname: Madhuprakash
authors.forename: Karunakar
authors.initials: K
authors.lastname: Tanneeru
authors.forename: Pallinti
authors.initials: P
authors.lastname: Purushotham
authors.forename: Lalitha
authors.initials: L
authors.lastname: Guruprasad
authors.forename: Appa Rao
authors.initials: AR
authors.lastname: Podile
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002688:Chitinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D009844:Oligosaccharides / Q000378:metabolism; D011485:Protein Binding; D012705:Serratia / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D009844:Oligosaccharides; D002688:Chitinases