title: Biochemical and structural characterization of a novel bacterial manganese-dependent hydroxynitrile lyase.
abstract: Hydroxynitrile lyases (HNLs), which catalyse the decomposition of cyanohydrins, are found mainly in plants. In vitro, they are able to catalyse the synthesis of enantiopure cyanohydrins, which are versatile building blocks in the chemical industry. Recently, HNLs have also been discovered in bacteria. Here, we report on the detailed biochemical and structural characterization of a hydroxynitrile lyase from Granulicella tundricola (GtHNL), which was successfully heterologously expressed in Escherichia coli. The crystal structure was solved at a crystallographic resolution of 2.5 Å and exhibits a cupin fold. As GtHNL does not show any sequence or structural similarity to any other HNL and does not contain conserved motifs typical of HNLs, cupins represent a new class of HNLs. GtHNL is metal-dependent, as confirmed by inductively coupled plasma/optical emission spectroscopy, and in the crystal structure, manganese is bound to three histidine and one glutamine residue. GtHNL displayed a specific activity of 1.74 U·mg(-1) at pH 6 with (R)-mandelonitrile, and synthesized (R)-mandelonitrile with 90% enantiomeric excess at 80% conversion using 0.5 m benzaldehyde in a biphasic reaction system with methyl tertiary butyl ether.
authors: Austrian Centre of Industrial Biotechnology GmbH, Graz, Austria; Gebouw voor Scheikunde, Technische Universiteit Delft, The Netherlands.
authors: Ivan
authors: I
authors: Hajnal
authors: Andrzej
authors: A
authors: Lyskowski
authors: Ulf
authors: U
authors: Hanefeld
authors: Karl
authors: K
authors: Gruber
authors: Helmut
authors: H
authors: Schwab
authors: Kerstin
authors: K
authors: Steiner
mesh_terms: D000097:Acetonitriles / Q000737:chemistry / Q000378:metabolism; D061271:Acidobacteria / Q000201:enzymology* / Q000235:genetics; D000446:Aldehyde-Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020816:Amino Acid Motifs; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain / Q000235:genetics; D018360:Crystallography, X-Ray; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008345:Manganese / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013237:Stereoisomerism
chemical_list: D000097:Acetonitriles; D001426:Bacterial Proteins; D011994:Recombinant Proteins; D008345:Manganese; C045516:mandelonitrile; D000446:Aldehyde-Lyases; C021541:mandelonitrile lyase
keywords: bacterial hydroxynitrile lyase; cupin structure; enzyme catalysis; metalloenzyme; site-directed mutagenesis