EPR Study of Substrate Binding to Mn(II) in Hydroxynitrile Lyase from Granulicella tundricola

Abstract

GtHNL from Granulicella tundricola is a Mn­(II) containing hydroxynitrile lyase with a cupin fold. The quasi-octahedral manganese is pentacoordinated by the enzyme. It catalyzes the enantioselective addition of HCN to aldehydes, yielding R-cyanohydrins. On the Lewis acidic vacant coordination site the Mn binds either substrate or the product, leading to a hexacoordinated 17 electron complex. EPR spectra of the active enzyme are unusually wide with a zero-field splitting approximately equal to the X-band microwave energy. A spectral change is induced by incubation with either one of the substrates/products HCN, benzaldehyde, and/or mandelonitrile. This points toward Mn­(II) catalyzed cyanohydrin synthesis.

CH‐π Interactions Promote the Conversion of Hydroxypyruvate in a Class II Pyruvate Aldolase

“…These results are of particular interest, since solvated Mg 2+ and Mn 2+ ions are known to be poor Lewis acid catalysts in aqueous media . A previous study on the Mn 2+ containing enzyme Gt HNL suggested a distorted octahedral coordination geometry to be responsible for the increased Lewis acidity, where the connecting line between the two tops of the square‐based pyramid deviates from the expected angle of 180° . A similar deviation was also observed in the crystal structure of holo‐ Sw HKA for the Mg 2+ cluster (6r62.pdb).…”

“…A similar change in the zero‐field splitting has previously been reported for the Mn(II) bound extradiol cleaving catechol dioxygenase upon the addition of the substrate 3,4‐dihydroxyphenylacetate and for Mn(II) bound pyruvate kinase upon the addition of pyruvate or phosphoenolpyruvate . Spectra similar to the pyruvate bound Mn‐ Sw HKA have also been reported for oxalate decarboxylase, oxalate oxidase, hydroxynitrile lyase Gt HNL and phosphoglucosemutase …”

“…Additional lines result from semi‐forbidden transitions that have a lower intensity. A more extensive description of Mn 2+ EPR of proteins has been published previously . The EPR spectrum of Mn 2+ bound aldolase is dominated by the characteristic six line pattern around g=2, with a hyperfine coupling constant of circa 95 Gauss.…”

The crystal structure and catalytic mechanism of hydroxynitrile lyase from passion fruit, Passiflora edulis

“…H8A mutant was completely inactive, whereas Y30F mutant exhibited a low activity (~ 5% of wild‐type). Thus, His8 is essential for the reaction and acts as a general base to deprotonate the hydroxyl group in Pe HNL as observed in other HNLs []. The mutants of Asn101 (N101A and N101D) were completely inactive.…”

“…HNLs include diverse groups of enzymes belonging to various protein families. Six unrelated structural classes of HNLs have been reported so far; glucose‐methanol‐choline oxidoreductase , α/β‐hydrolase , carboxypeptidase , zinc‐dependent alcohol dehydrogenase , cupin superfamily , and recently solved Bet v1 superfamily . Although these HNLs react with similar or identical substrates, the architectures of their active sites are largely different.…”