Mustak A. Kaderbhai

Biodiversity of the P450 catalytic cycle: yeast cytochrome b5/NADH cytochrome b5 reductase complex efficiently drives the entire sterol 14-demethylation (CYP51) reaction.

The widely accepted catalytic cycle of cytochromes P450 (CYP) involves the electron transfer from NADPH cytochrome P450 reductase (CPR), with a potential for second electron donation from the microsomal cytochrome b 5/NADH cytochrome b 5 reductase system. The latter system only supported CYP reactions inefficiently. Using purified proteins including Candida albicans CYP51 and yeast NADPH cytochrome P450 reductase, cytochrome b 5 and NADH cytochrome b 5 reductase, we show here that fungal CYP51 mediated sterol 14α‐demethylation can be wholly and efficiently supported by the cytochrome b 5/NADH cytochrome b 5 reductase electron transport system. This alternative catalytic cycle, where both the first and second electrons were donated via the NADH cytochrome b 5 electron transport system, can account for the continued ergosterol production seen in yeast strains containing a disruption of the gene encoding CPR.

Minipreps of plasmid DNA.

Although there are a large number of protocols for the isolation of small quantities of plasmid DNA from bacterial cells (minipreps), this unit presents four procedures based on their speed and success: the alkaline lysis prep, a modification of the alkaline lysis prep that is performed in 1.5‐ml tubes or 96‐well microtiter dishes, the boiling method, and a lithium‐based procedure. A support protocol provides information on storing plasmid DNA.

A photoactivatable synthetic transit peptide labels 30 kDa and 52 kDa polypeptides of the chloroplast inner envelope membrane

A 24‐residue transit peptide based on the sequence of a precursor of the small subunit of wheat ribulose‐1,5‐bisphosphate carboxylase (Rubisco) was synthesized. The transit peptide was converted into a radioactive azido derivatised analogue. Photoactivation of the radiolabelled transit peptide analogue with isolated inner and outer membranes of the chloroplast envelope intensely labelled two proteins of 30 kDa and 52 kDa. In the outer membrane only the 52 kDa polypeptide was labelled. These findings are consistent with a recent report on the identification of the 30 kDa receptor protein for protein import in the chloroplast envelope contact zones [(1988) Nature 331, 232–237].

Rapid and quantitative analysis of recombinant protein expression using pyrolysis mass spectrometry and artificial neural networks: application to mammalian cytochrome b5 in Escherichia coli

Recombinant Escherichia coli clones encoding between 0 and 6 copies of the mammalian cytochrome b5 gene were subjected to pyrolysis mass spectrometry (PyMS). To deconvolute the pyrolysis mass spectra so as to obtain quantitative information on the amount of cytochrome b5 produced fully-interconnected feedforward artificial neural networks (ANNs) were studied. It was found that the combination of PyMS and ANNs could be used to predict the amount of cytochrome b5 expressed in E. coli. PyMS is a novel, convenient and rapid method for the screening and analysis of microbial and other cultures producing recombinant proteins.

Export of Cytochrome P450 105D1 to the Periplasmic Space of Escherichia coli

ABSTRACT CYP105D1, a cytochrome P450 from Streptomyces griseus, was appended at its amino terminus to the secretory signal of Escherichia coli alkaline phosphatase and placed under the transcriptional control of the nativephoA promoter. Heterologous expression in E. coli phosphate-limited medium resulted in abundant synthesis of recombinant CYP105D1 that was translocated across the bacterial inner membrane and processed to yield authentic, heme-incorporated P450 within the periplasmic space. Cell extract and whole-cell activity studies showed that the periplasmically located CYP105D1 competently catalyzed NADH-dependent oxidation of the xenobiotic compounds benzo[a]pyrene and erythromycin, further revealing the presence in the E. coli periplasm of endogenous functional redox partners. This system offers substantial advantages for the application of P450 enzymes to whole-cell biotransformation strategies, where the ability of cells to take up substrates or discard products may be limited.

Gene-dose-dependent expression of soluble mammalian cytochrome b5 in Escherichia coli

SummaryA synthetic structural gene encoding a mammalian cytochrome b5, carrying an optimised ribosomal binding sequence, was tandemly polymerised ranging from one (n=1) to six (n=6) gene copies. The gene, placed in pλ-ncyt under the control of the λPL promoter, transcribed mono- to hexahomocistronic mRNA, expressing one to six copies of cytochrome b5. The expressed levels of cytochrome b5 in Escherichia coli pλ-ncyt corresponded linearly with the gene dose when up to five copies were present; saturating build-up of the recombinant protein was reached at six gene copies. Cells bearing pλ-6cyt produced 75 μg cytochrome b5/ml of unit optical density at 600 nm culture, constituting 55% of the soluble bacterial protein. The recombinant protein accumulated predominantly in a haem-deficient, apoform, together with lesser amounts of the halocytochrome b5. Whereas the overall expressed protein (apo and holo forms) was gene dose dependent, there was an inverse relationship between holocytochrome b5 production and gene dose. Incubation of the thermally induced bacterial lysates with exogenous haem a converted all of the soluble apocytochrome b5 into holocytochrome b5 that was spectrally indistinguishable with its native counterpart. Culture supplementation with the likely metabolic precursors of haem synthesis, 5-aminolevulinic acid, glycine/succinate or glutamate, significantly alleviated the protoporphyrin deficiency during hyperproduction of cytochrome b5 in E. coli.

A directed evolution strategy for optimized export of recombinant proteins reveals critical determinants for preprotein discharge.

A directed evolutionary approach is described that searches short, random peptide sequences for appendage at the secretory signal peptide–mature protein junction to seek ideal algorithms for both efficient and hyper export of recombinant proteins to the periplasm of Escherichia coli. The strategy employs simple, visual detection of positive clones using a PINK expression system that faithfully reports on export status of a mammalian hemoprotein in E. coli. With‐in “sequence spaces” ranging from 1 to 13 residues, a significant but highly variable secretory fitness was scored such that the rate of secretion reciprocally correlated with the membrane‐associated precursor pool of the evolved exportable hemoproteins. Three clusters of hyper, median, and hypo exporters were isolated. These had corresponding net charges of −1, 0, and +1 within the evolved sequence space, which in turn clearly correlated with the prevailing magnitude and polarity of the membrane energization states. The findings suggest that both the nature of the charged residue and the proximal sequence in the early mature region are the crucial determinants of the protonophore‐dependent electrophoretic discharge of the precursor across the inner membrane of E. coli. We conclude that the directed evolutionary approach will find ready application in engineering recombinant proteins for their efficient secretion via the sec export pathway in E. coli.

An improved and rapid procedure for isolating RNA-Free Escherichia coli plasmid DNA

We describe a simple, rapid, and inexpensive procedure for the isolation of plasmid DNA in high yields from Escherichia coli cultures. The procedure entails two main steps, which involve treating intact bacterial cells with phenol/chloroform in the presence of Triton X-100 and LiCl followed by polyethylene glycol precipitation. Plasmid DNA preparations isolated by this method are highly pure and virtually devoid of RNA. The DNA is suitable substrate for restriction mapping, DNA-modifying enzymes, and in vitro transcription with SP6 and T7 RNA polymerases.

The quinohaemoprotein lupanine hydroxylase from Pseudomonas putida

Lupanine hydroxylase catalyses the first reaction in the catabolism of the alkaloid lupanine by Pseudomonas putida. It dehydrogenates the substrate, which can then be hydrated. It is a monomeric protein of M(r) 72,000 and contains a covalently bound haem and a molecule of PQQ. The gene for this enzyme has been cloned and sequenced and the derived protein sequence has a 26 amino acid signal sequence at the N-terminal for translocation of the protein to the periplasm. Many of the features seen in the sequence of lupanine hydroxylase are common with other quinoproteins including the W-motifs that are characteristic of the eight-bladed propeller structure of methanol dehydrogenase. However, the unusual disulfide bridge between adjacent cysteines that is present in some PQQ-containing enzymes is absent in lupanine hydroxylase. The C-terminal domain contains characteristics of a cytochrome c and overall the sequence shows similarities with that of the quinohaemoprotein, alcohol dehydrogenase from Comamonas testosteroni. The gene coding for lupanine hydroxylase has been successfully expressed in Escherichia coli and a procedure has been developed to renature and reactivate the enzyme, which was found to be associated with the inclusion bodies. Reactivation required addition of PQQ and was dependent on calcium ions.

Kinetic constants of signal peptidase I using cytochrome b5 as a precursor substrate.

A procedure is described for measuring Escherichia coli signal peptidase I activity which exploits an intact precursor protein composed of the alkaline phosphatase signal peptide fused to the full length mammalian cytochrome b5. This cytochrome b5 precursor protein has been extensively characterised and shown to be processed accurately by purified signal peptidase I [Protein Expr. Purif. 7 (1996) 237]. The amphipathic, chimaeric cytochrome b5 precursor was isolated in mg quantities in a highly homogeneous state under non-denaturing conditions. The processing of the cytochrome b5 precursor by signal peptidase displayed Michaelis-Menten kinetics with K(m)=50 microM and k(cat)=11 s(-1). The K(m) was 20-fold lower than that obtained with signal peptide substrates and 3-fold higher than that reported for pro-OmpA-nuclease A precursor fusion. The corresponding turnover number, k(cat), was four orders of magnitude greater than the peptide substrates but was 2-fold lower than pro-OmpA-nuclease A precursor fusion. These results confirm that both the affinities and the catalytic power of the signal peptidase are significantly higher for macromolecular precursor substrates than for the shorter signal peptide substrates.