title: Prokaryotic squalene-hopene cyclases can be converted to citronellal cyclases by single amino acid exchange.
abstract: Squalene-hopene cyclases (SHCs) are prokaryotic enzymes that catalyse the cyclisation of the linear precursor squalene to pentacyclic hopene. Recently, we discovered that a SHC cloned from Zymomonas mobilis (ZMO-1548 gene product) has the unique property to cyclise the monoterpenoid citronellal to isopulegol. In this study, we performed saturation mutagenesis of three amino acids of the catalytic centre of ZMO-1548 (F428, F486 and W555), which had been previously identified to interact with enzyme-bound substrate. Replacement of F428 by tyrosine increased hopene formation from squalene, but isopulegol-forming activity was strongly reduced or abolished in all muteins of position 428. W555 was essential for hopene formation; however, three muteins (W555Y, W428F or W555T) revealed enhanced cyclisation efficiency with citronellal. The residue at position 486 turned out to be the most important for isopulegol-forming activity. While the presence of phenylalanine or tyrosine favoured cyclisation activity with squalene, several small and/or hydrophobic residues such as cysteine, alanine or isoleucine and others reduced activity with squalene but greatly enhanced isopulegol formation from citronellal. Replacement of the conserved aromatic residue corresponding to F486 to cysteine in other SHCs cloned from Z. mobilis (ZMO-0872), Alicyclobacillus acidocaldarius (SHC(Aac)), Acetobacter pasteurianus (SHC(Apa)), Streptomyces coelicolor (SHC(Sco)) and Bradyrhizobium japonicum (SHC(Bja)) resulted in more or less strong isopulegol-forming activities from citronellal. In conclusion, many SHCs can be converted to citronellal cyclases by mutagenesis of the active centre thus broadening the applicability of this interesting class of biocatalyst.
authors.affiliation: Institute of Microbiology, University of Stuttgart, Allmandring 31, 70550 Stuttgart, Germany.
authors.forename: Gabriele
authors.initials: G
authors.lastname: Siedenburg
authors.forename: Michael
authors.initials: M
authors.lastname: Breuer
authors.forename: Dieter
authors.initials: D
authors.lastname: Jendrossek
mesh_terms: D000080462:Acyclic Monoterpenes; D000262:Adenylyl Cyclases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000447:Aldehydes / Q000378:metabolism*; D019943:Amino Acid Substitution; D001419:Bacteria / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D019751:Intramolecular Transferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D039821:Monoterpenes / Q000378:metabolism*; D020125:Mutation, Missense; D013379:Substrate Specificity; D017355:Zymomonas / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
chemical_list: D000080462:Acyclic Monoterpenes; D000447:Aldehydes; D001426:Bacterial Proteins; D039821:Monoterpenes; D000262:Adenylyl Cyclases; D019751:Intramolecular Transferases; C066630:squalene-hopene cyclase; C108217:citronellal