First purified recombinant CYP75B including transmembrane helix with unexpected high substrate specificity to (2R)-naringenin.

Anthochlor pigments (chalcones and aurones) play an important role in yellow flower colourization, the formation of UV-honey guides and show numerous health benefits. The B-ring hydroxylation of chalcones is performed by membrane bound cytochrome P450 enzymes. It was assumed that usual flavonoid 3'-hydroxlases (F3'Hs) are responsible for the 3,4- dihydroxy pattern of chalcones, however, we previously showed that a specialized F3'H, namely chalcone 3-hydroxylase (CH3H), is necessary for the hydroxylation of chalcones. In this study, a sequence encoding membrane bound CH3H from Dahlia variabilis was recombinantly expressed in yeast and a purification procedure was developed. The optimized purification procedure led to an overall recovery of 30% recombinant DvCH3H with a purity of more than 84%. The enzyme was biochemically characterized with regard to its kinetic parameters on various substrates, including racemic naringenin, as well as its enantiomers (2S)-, and (2R)-naringenin, apigenin and kaempferol. We report for the first time the characterization of a purified Cytochrome P450 enzyme from the flavonoid biosynthesis pathway, including the transmembrane helix. Further, we show for the first time that recombinant DvCH3H displays a higher affinity for (2R)-naringenin than for (2S)-naringenin, although (2R)-flavanones are not naturally formed by chalcone isomerase.

Molecular and Enzymatic Characterization of Flavonoid 3'-Hydroxylase of Malus × domestica.

Malus × domestica (apple) accumulates particularly high amounts of dihydrochalcones in various tissues, with phloridzin (phloretin 2'-O-glucoside) being prevalent, although small amounts of 3-hydroxyphloretin and 3-hydroxyphloridzin are also constitutively present. The latter was shown to correlate with increased disease resistance of transgenic M. × domestica plants. Two types of enzymes could be involved in 3-hydroxylation of dihydrochalcones: polyphenol oxidases or the flavonoid 3'-hydroxylase (F3'H), which catalyzes B-ring hydroxylation of flavonoids. We isolated two F3'H cDNA clones from apple leaves and tested recombinant Malus F3'Hs for their substrate specificity. From the two isolated cDNA clones, only F3'HII encoded a functionally active enzyme. In the F3'HI sequence, we identified two putatively relevant amino acids that were exchanged in comparison to that of a previously published F3'HI. Site directed mutagenesis, which exchanged an isoleucine into methionine in position 211 restored the functional activity, which is probably because it is located in an area involved in interaction with the substrate. In contrast to high activity with various flavonoid substrates, the recombinant enzymes did not accept phloretin under assay conditions, making an involvement in the dihydrochalcone biosynthesis unlikely.

Recombinant production of a hard-to-express membrane-bound cytochrome P450 in different yeasts-Comparison of physiology and productivity.

Cytochrome P450s comprise one of the largest protein superfamilies. They occur in every kingdom of life and catalyse a variety of essential reactions. Their production is of utmost interest regarding biotransformation and structure-function elucidation. However, they have proven hard to express due to their membrane anchor, their complex co-factor requirements and their need for a redox-partner. In our study, we investigated and compared different yeast strains for the production of the plant cytochrome P450 chalcone 3-hydroxylase. To our knowledge, this is the first study evaluating different yeasts for the expression of this abundant and highly significant protein superfamily. Saccharomyces cerevisiae and three different strains of Pichia pastoris expressing chalcone 3-hydroxylase were cultivated in controlled bioreactor runs and evaluated regarding physiological parameters and expression levels of the cytochrome P450. Production differed significantly between the different strains and was found highest in the investigated P. pastoris MutS strain KM71H where 8 mg P450 per gram dry cell weight were detected. We believe that this host could be suitable for the expression of many eukaryotic, especially plant-derived, cytochrome P450s as it combines high specific product yields together with straightforward cultivation techniques for achieving high biomass concentrations. Both factors greatly facilitate subsequent establishment of purification procedures for the cytochrome P450 and make the yeast strain an ideal platform for biotransformation as well.

E. coli HMS174(DE3) is a sustainable alternative to BL21(DE3).

BACKGROUND: Escherichia coli is one of the most widely used hosts for recombinant protein production in academia and industry. Strain BL21(DE3) is frequently employed due to its advantageous feature of lacking proteases which avoids degradation of target protein. Usually it is used in combination with the T7-pET system where induction is performed by one point addition of IPTG. We recently published a few studies regarding lactose induction in BL21(DE3) strains. BL21(DE3) can only take up the glucose-part of the disaccharide when fed with lactose. However, initially additional glucose has to be supplied as otherwise the ATP-related lactose uptake barely happens. Yet, as lactose is an inexpensive compound compared to glucose and IPTG, a new induction strategy by a lactose-only feed during induction seems attractive. Thus, we investigated this idea in the galactose metabolizing strain HMS174(DE3). RESULTS: We show that strain HMS174(DE3) can be cultivated on lactose as sole carbon source during induction. We demonstrate that strain HMS174(DE3) exhibits higher product and biomass yields compared to BL21(DE3) when cultivated in a lactose fed-batch. More importantly, HMS174(DE3) cultivated on lactose even expresses more product than BL21(DE3) in a standard IPTG induced glucose fed-batch at the same growth rate. Finally, we demonstrate that productivity in HMS174(DE3) lactose-fed batch cultivations can easily be influenced by the specific lactose uptake rate (qs,lac). This is shown for two model proteins, one expressed in soluble form and one as inclusion body. CONCLUSIONS: As strain HMS174(DE3) expresses even slightly higher amounts of target protein in a lactose fed-batch than BL21(DE3) in a standard cultivation, it seems a striking alternative for recombinant protein production. Especially for large scale production of industrial enzymes cheap substrates are essential. Besides cost factors, the strategy allows straight forward adjustment of specific product titers by variation of the lactose feed rate.

Posttranslational Modification of Heme b in a Bacterial Peroxidase: The Role of Heme to Protein Ester Bonds in Ligand Binding and Catalysis.

The existence of covalent heme to protein bonds is the most striking structural feature of mammalian peroxidases, including myeloperoxidase and lactoperoxidase (LPO). These autocatalytic posttranslational modifications (PTMs) were shown to strongly influence the biophysical and biochemical properties of these oxidoreductases. Recently, we reported the occurrence of stable LPO-like counterparts with two heme to protein ester linkages in bacteria. This study focuses on the model wild-type peroxidase from the cyanobacterium Lyngbya sp. PCC 8106 (LspPOX) and the mutants D109A, E238A, and D109A/E238A that could be recombinantly produced as apoproteins in Escherichia coli, fully reconstituted to the respective heme b proteins, and posttranslationally modified by hydrogen peroxide. This for the first time allows not only a direct comparison of the catalytic properties of the heme b and PTM forms but also a study of the impact of D109 and E238 on PTM and catalysis, including Compound I formation and the two-electron reduction of Compound I by bromide, iodide, and thiocyanate. It is demonstrated that both heme to protein ester bonds can form independently and that elimination of E238, in contrast to exchange of D109, does not cause significant structural rearrangements or changes in the catalytic properties neither in heme b nor in the PTM form. The obtained findings are discussed with respect to published structural and functional data of human peroxidases.