Truncation of N-terminal regions of Digitalis lanata progesterone 5β-reductase alters catalytic efficiency and substrate preference

Characteristics of thermostable amylopullulanase of Geobacillus thermoleovorans and its truncated variants

“…The K m value of gt-apu N showed a significant decrease with an increased V max and k cat for pullulan as compared to gt-apu and gt-apu C. This is contrary to the observations recorded for the ␣-amylase activity as suggested from a higher K m and a lower k cat values for soluble starch than gt-apu and gt-apu C. A decreased K m with high turnover (k cat ) values for pullulan had also been reported for ␣-amylase 1 of T. vulgaris R-47 by deleting 11 consecutive amino acids and mutagenesis [21]. Rudolph et al [22] and Lu et al [23] had also observed recently an altered catalytic efficiency and substrate preference for the N-terminal truncated Digitalis lanata progesterone 5␤-reductase. This suggests that the N-terminal truncation might have changed the structure of the substrate binding site to a stable conformation which favours pullulan over starch.…”

Expression, crystallization and structure elucidation of γ-terpinene synthase fromThymus vulgaris

“…Polymerase-chain-reaction amplifications were performed with Phusion High-Fidelity DNA Polymerase (New England BioLabs, Ipswich, England) as described previously (Rudolph et al, 2014). A peqSTAR 96 Universal Gradient thermocycler (PEQLAB Biotech GmbH, Erlangen, Germany) was used for amplification according to the supplier's recommendation.…”

“…Expression-vector constructs were created using the vectors pENTR-D-TOPO and Champion pET300/NT-DEST (Invitrogen, Karlsruhe, Germany), providing a hexahistidine tag (His 6 ) at the N-terminus of TvTPS (Table 1). To generate the single-amino-acid exchange variants of TvTPS1 (D356G and T565A), site-directed mutagenesis was carried out as described by Bauer et al (2012) and Rudolph et al (2014).…”

Conversion of Substrate Analogs Suggests a Michael Cyclization in Iridoid Biosynthesis

“…For these studies, an enzyme with a truncation at the N terminus was used, which increases the structural stability of the nearest iridoid synthase homolog, progesterone beta-reductase. This truncation has recently been shown to affect kinetic parameters for progesterone beta-reductase ( Rudolph et al., 2014 ). Therefore, kinetic parameters for 2 were remeasured using this truncated enzyme.…”