Oxidative cleavage of alkenes is a widely employed process allowing oxyfunctionalization to corresponding carbonyl compounds. Recently, a novel biocatalytic oxidative alkene cleavage activity on styrene derivatives was identified in TM1459 from Thermotoga maritima. In this work we engineered the enzyme by site-saturation mutagenesis of active site amino acids to increase its activity and to broaden its substrate scope. A high-throughput assay for the detection of the ketone products was successfully developed. Several variants with up to twofold improved conversion level of styrene derivatives were successfully identified. Especially, changes in or removal of the C-terminus of TM1459 increased the activity most significantly. These best variants also displayed a slightly enlarged substrate scope.
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.
In conjugative elements such as integrating conjugative elements (ICEs) or conjugative plasmids (CPs) transcription of DNA transfer genes is a prerequisite for cells to become transfer competent, i.e., capable of delivering plasmid DNA via bacterial conjugation into new host bacteria. In the large family of F-like plasmids belonging to the MobF 12 A group, transcription of DNA transfer genes is tightly controlled and dependent on the activation of a single promoter, designated P Y. Plasmid encoded TraJ and chromosomally encoded ArcA proteins are known activators, whereas the nucleoid associated protein heat-stable nucleoid structuring (H-NS) silences the P Y promoter. To better understand the role of these proteins in P Y promoter activation, we performed in vitro DNA binding studies using purified H-NS, ArcA, and TraJ R1 (TraJ encoded by the conjugative resistance plasmid R1). All proteins could bind to R1P Y DNA with high affinities; however, only ArcA was found to be highly sequence specific. DNase I footprinting studies revealed three H-NS binding sites, confirmed the binding site for ArcA, and suggested that TraJ contacts a dyad symmetry DNA sequence located between −51 and −38 in the R1P Y promoter region. Moreover, TraJ R1 and ArcA supplied together changed the H-NS specific protection pattern suggesting that these proteins are able to replace H-NS from R1P Y regions proximal to the transcription start site. Our findings were corroborated by P Y-lacZ reporter fusions with a series of site specific R1P Y promoter mutations. Sequential changes of some critical DNA bases in the TraJ binding site (jbs) from plasmid R1 to plasmid F led to a remarkable specificity switch: The P Y promoter became activatable by F encoded TraJ whereas TraJ R1 lost its activation function. The R1P Y mutagenesis approach also confirmed the requirement for the host-encoded response-regulator ArcA and indicated that the sequence context, especially in the −35 region is critical for P Y regulation and function.
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