title: A complete library of amino acid alterations at R306 in Streptomyces clavuligerus deacetoxycephalosporin C synthase demonstrates its structural role in the ring-expansion activity.
abstract: In a previous study, the conserved arginine residue at position 306 of Streptomyces clavuligerus deacetoxycephalsoporin C synthase (scDAOCS), when mutated to leucine, resulted in 191% increase in converting ampicillin to its expanded cephalosporin moiety compared with that of the wild-type enzyme. However, the role of this residue in eliciting the improved enzymatic activity is not well understood. In this study, probing the molecular basis of amino acid substitutions at position 306 has underscored its importance for engineering various improvements in the ring expansion activity. Structural modeling using SwissPdbViewer revealed that R306 is surrounded by a hydrophobic cleft formed by residues Y184, L186, W297, I298, and V303. Hence, the improved activity achieved by the R306L mutation was probably because of better hydrophobic packing in this region. To evaluate the role of amino acids at position 306 of scDAOCS and its influence on the molecular status of the enzyme at this locality, alteration to 18 other amino acids was done by site-directed mutagenesis. The effects of each substitution on the enzyme activity were determined by bioassay using penicillin substrates: ampicillin, penicillin G, phenethicillin, and carbenicillin. Results obtained showed a drastic reduction in enzyme activity when R306 was replaced with charged or polar residues, thus emphasizing the importance of hydrophobic packing around this site. The bioassay results also illustrated that apart from leucine, substitutions to nonpolar residues, isoleucine and methionine, were able to improve the ampicillin conversion activity of scDAOCS by 168 and 113% of the wild-type enzyme activity, respectively. Similar trend of effects from each mutation was also observed for penicillin G, phenethicillin, and carbenicillin conversions. The enhanced enzyme activities were supported by spectrophotometric assay indicating that all these mutants have lower K(m) values (R306L: 1.09 mM; R306I: 2.64 mM; R306M: 5.68 mM) than the wild-type enzyme (8.33 mM), resulting in improvement in the enzyme's substrate binding affinity. Hence, this mutational study of amino acids situated at 306 of scDAOCS has provided a better understanding of the significance of specific amino acid residues at this position which can improve its ring-expansion activity when given a plethora of beta-lactam substrates to generate corresponding, possibly new, cephalosporins.
authors.affiliation: Department of Microbiology, Yong Loo Lin School of Medicine, Singapore 117597.
authors.forename: Kian
authors.initials: K
authors.lastname: Sim Goo
authors.forename: Chun
authors.initials: C
authors.lastname: Song Chua
authors.forename: Tiow-Suan
authors.initials: TS
authors.lastname: Sim
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002384:Catalysis; D057927:Hydrophobic and Hydrophilic Interactions; D019751:Intramolecular Transferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D046915:Penicillin-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D013302:Streptomyces / Q000201:enzymology*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D046915:Penicillin-Binding Proteins; D011994:Recombinant Proteins; D019751:Intramolecular Transferases; C038371:deacetoxycephalosporin C synthetase