PubMed Health⌕ Search

Biomedical subjects

M A Kaderbhai

Publications and source records attributed to M A Kaderbhai.

At least 19 recordsLinked to original sources
PubMed2006

Towards engineered topogenesis of cytochrome b(5) and P450 for in vivo transformation of xenobiotics.

Nature is endowed with catalysts capable of an unprecedented diversity of biotransformations, beyond the capabilities of synthetic chemistries. In a biotechnological context, there is a growing and emerging need to tap this catalytic potential. CYP (cytochrome P450) represents a superfamily of enzymes capable of a diverse array of catalytic activities. Distinct members are engaged in biosynthetic reactions within many organisms, while others have a role in the detoxification of foreign compounds. The latter substrates include medicines, pollutants, pesticides, carcinogens, perfumes and herbicides, representing considerable applied importance for pharmacology and toxicology. CYPs show a high degree of stereo- and regio-specificity for their reactions, which have wide industrial applications. Recombinant CYPs are commonly expressed as active recombinant cytosolic forms in Escherichia coli. However, selective permeability of E. coli to many substrates and products can cause problems with product recovery when using whole-cell systems. To overcome these problems, we have been developing approaches to facilitate export of functional recombinant haemoproteins to the inner membrane, periplasm and the outer membrane of E. coli. Here, we describe the progress in relation to cytochrome b(5) and CYPs.

Biotransformation↗
PubMed2005

Cytochrome b(5) modulation of 17{alpha} hydroxylase and 17-20 lyase (CYP17) activities in steroidogenesis.

CYP17 is a steroidogenic enzyme located in the zona fasciculata and zona reticularis of the adrenal cortex and gonad tissues and which has dual functions - hydroxylation and as a lyase. The first activity gives hydroxylation of pregnenolone and progesterone at the C(17) position to generate 17alpha-hydroxypregnenolone and 17alpha-hydroxyprogesterone, while the second enzymic activity cleaves the C(17)-C(20) bond of 17alpha-hydroxypregnenolone and 17alpha-hydroxyprogesterone to form dehydroepiandro-sterone and androstenedione respectively. The modulation of these two activities occurs through cytochrome b(5). Association of cytochrome b(5) and CYP17 is thought to be based primarily on electrostatic interactions in which the negatively charged residues pair up with positively charged residues on the proximal surface of the CYP17 molecule. Non-specific interactions of the hydrophobic membrane regions of cytochrome b(5) and CYP17 are also thought to play a crucial role in the association of these two haemoproteins. Although cytochrome b(5) is known to stimulate CYP activity by contributing the second electron in the catalytic cycle, in the case of CYP17, the mechanism of cleavage stimulation proceeds via an allosteric mode. It is hypothesised that cytochrome b(5) promotes the cleavage by aligning the iron-oxygen complex attack onto the C(20) rather than the C(17) atom of the steroid substrate molecule. Thus, further understanding of the mechanism of modulation by cytochrome b(5) of the hydroxylase and lyase activities should shed new insights on developing therapeutic targets in CYP17-linked biochemical processes such as adrenarche, polycystic ovary syndrome and prostate cancer.

Adrenal Cortex↗
PubMed2001

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.

Alkaline Phosphatase↗
PubMed2001

Human sterol 14alpha-demethylase activity is enhanced by the membrane-bound state of cytochrome b(5).

Human sterol 14alpha-demethylase (P45051; CYP51) catalyzes the oxidative removal of the C32 methyl group of dihydrolanosterol, an essential step in the cholesterol biosynthetic pathway. The reaction is dependent upon NADPH cytochrome P450 reductase (CPR) that donates the electrons for the catalytic cycle. Here we used a recombinant yeast CPR to investigate the abilities of four different forms of cytochrome b(5) to support sterol demethylation activity of CYP51. The cytochrome b(5) derivatives were genetically engineered forms of the native rat cytochrome b(5) core-tail: the soluble globular b(5) core (core), the core linked at its N-terminus with the secretory signal sequence of alkaline phosphatase (signal-core), and the signal sequence linked to the native b(5) (signal-core-tail). The rat core-tail enzyme greatly stimulated sterol demethylation, whereas the signal-core-tail was only marginally active. In contrast, the core and signal-core constructs were completely inactive in stimulating the demethylation reaction. Additionally, cytochrome b(5) enhanced sterol demethylation by more than threefold by accepting electrons from soluble yeast CPR and in its ability to reduce P450. We show that the nature of transient linkage between the hemoproteins and the redox partners is most likely brought about electrostatically, although productive interaction between cytochrome b(5) and CYP51 is governed by the membrane-insertable hydrophobic region in the cytochrome b(5) which in turn determines the correct spatial orientation of the core. This is the first report showing the stimulation of CYP51 by cytochrome b(5).

Amino Acid Sequence↗
PubMed2001

Export of cytochrome P450 105D1 to the periplasmic space of Escherichia coli.

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 native phoA 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.

Bacterial Proteins↗
PubMed2000

Targeting of active human cytochrome P4501A1 (CYP1A1) to the periplasmic space of Escherichia coli.

Native human cytochrome P4501A1 (CYP1A1) was appended at its amino terminus to the secretory signal of Escherichia coli alkaline phosphatase. The chimeric P450 construct was placed under the transcriptional control of the native phoA promoter in a prokaryotic expression vector. Induction of the hemoprotein by heterologous expression in E. coli following growth in a phosphate-limited medium resulted in abundant synthesis of recombinant CYP1A1 as detected by reduced CO-difference spectra. Furthermore, the signal-appended CYP1A1 was translocated across the bacterial inner membrane by the sec-dependent pathway and processed to yield authentic, heme-incorporated P450 within the periplasmic space. In vitro and whole-cell metabolic activity studies showed that the periplasmically-located CYP1A1 competently catalysed NADPH-dependent benzo[a]pyrene 3-hydroxylation and 7-ethoxyresorufin O-deethylation. The means to localise cytochromes P450 in the periplasm offers an ability to produce high levels of protein, attributable to the less hostile nature of the compartment, and therein the enzymes for posttranslational assembly of heme with the translocated protein.

Alkaline Phosphatase↗
PubMed2000

A mammalian cytochrome fused to a chloroplast transit peptide is a functional haemoprotein and is imported into isolated chloroplasts.

The small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a major chloroplast stromal protein that is cytosolically synthesized as a precursor with an N-terminal extension, known as the transit sequence or transit peptide (Tp). The Tp is essential for the post-translational uptake of the precursor by the chloroplast. The Tp is thought to influence the conformation of the precursor protein and to facilitate polypeptide translocation across the chloroplast envelope barrier via a Tp-selective translocon. To address these issues we have devised a novel strategy to generate substrate amounts of a chloroplast targeting sequence as a fusion with the chromogenic globular domain of cytochrome b(5) (Cyt). The chimaeric protein is an ideal probe for investigating the conformation of a preprotein and events surrounding protein import into isolated chloroplasts. The Cyt of liver endoplasmic reticulum was fused at its N-terminus with the Tp of the small subunit of Rubisco of Pisum sativum (pea). To enhance its production by clearance from the cytoplasm of Escherichia coli, the chimaera was engineered by further N-terminal linkage of a prokaryotic secretory signal. Expression of this tripartite fusion resulted in mg quantities of the signal sequence-processed Tp-Cyt protein, which was eventually targeted to the membranes. The chromogenic nature of the chimaera and its localization to the bacterial membrane facilitated the biochemical isolation of the precursor in a soluble and functional form. The purified preprotein displayed spectral and enzymic properties that were indistinguishable from the native parental Cyt, implying an absence of observable influence of the Tp on the conformation of the haemoprotein. The chimaeric precursor was imported into the stroma of the isolated chloroplasts in a dose-dependent manner. Import was also strongly dependent upon exogenously supplied ATP. The stromally imported chimaeric precursor protein was processed to a size characteristic of Cyt.

Adenosine Triphosphate↗
PubMed2000

A chloroplast envelope-transfer sequence functions as an export signal in Escherichia coli.

The small subunit precursor of pea ribulose-1,5-bisphosphate carboxylase/oxygenase engineered with prokaryotic elements was expressed in Escherichia coli. This resulted in a dependable level of synthesis of the precursor protein in E. coli. The bacterially synthesised plant precursor protein was translocated from the cytoplasm and targeted to the outer membrane of the envelope zone. During the translocation step, a significant proportion of the precursor was processed to a soluble, mature SSU and found localised in the periplasm. The determined amino acid sequence of the isolated precursor showed that it had a deletion of an arginine residue at position -15 in the transit peptide. Expression of this transit peptide-appended mammalian cytochrome b(5) in E. coli displayed a targeting profile of the chromogenic chimera that was similar to that observed with the plant precursor protein.

Amino Acid Sequence↗
PubMed1999

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 b5/NADH cytochrome b5 reductase system. The latter system only supported CYP reactions inefficiently. Using purified proteins including Candida albicans CYP51 and yeast NADPH cytochrome P450 reductase, cytochrome b5 and NADH cytochrome b5 reductase, we show here that fungal CYP51 mediated sterol 14alpha-demethylation can be wholly and efficiently supported by the cytochrome b5/NADH cytochrome b5 reductase electron transport system. This alternative catalytic cycle, where both the first and second electrons were donated via the NADH cytochrome b5 electron transport system, can account for the continued ergosterol production seen in yeast strains containing a disruption of the gene encoding CPR.

Candida albicans↗
PubMed1999

2,4'-Dihydroxyacetophenone dioxygenase (EC 1.13.11.41) from Alcaligenes sp. 4HAP: a novel enzyme with an atypical dioxygenase sequence.

2,4'-Dihydroxyacetophenone dioxygenase (EC 1.13.11.41) was purified to homogeneity from Alcaligenes sp. 4HAP grown on 4-hydroxyacetophenone. Measurements of the M(r) of the native enzyme ranged from 81600 to 87000, whereas values of 21000 and 20379 were given by SDS/PAGE and electrospray MS respectively. The enzyme is a homotetramer and contains one atom of iron per molecule of enzyme. From C- and N-terminal analyses, primers for PCR were designed and the dad gene cloned and sequenced. The predicted amino acid sequence of dad, deduced from the nucleotide sequence, confirms the N-terminal amino acid sequencing data and contains the sequence of an internal tryptic peptide. It gave a calculated M(r) of 20364. The gene was expressed in Escherichia coli and yielded active enzyme. The derived amino acid sequence does not show significant similarity to other dioxygenases or any strong similarity to protein sequences presently available in the databases.

Acetophenones↗
PubMed1997

Interaction of human CYP17 (P-450(17alpha), 17alpha-hydroxylase-17,20-lyase) with cytochrome b5: importance of the orientation of the hydrophobic domain of cytochrome b5.

Human CYP17 (P-450(17alpha), 17alpha-hydroxylase-17,20-lyase)-catalysed side-chain cleavage of 17alpha-hydroxyprogestogens into androgens is greatly dependent on the presence of cytochrome b5. The native form of cytochrome b5 is composed of a globular core, residues 1-98, followed by a membrane insertable C-terminal tail, residues 99-133. In the present study the abilities of five different forms of cytochrome b5 to support the side-chain cleavage activity of CYP17 were compared. The five derivatives were: the native pig cytochrome b5 (native pig), its genetically engineered rat counterpart (core-tail), the soluble core form of the latter (core), the core with the secretory signal sequence of alkaline phosphatase appended to its N-terminal (signal-core) and the latter containing the C-terminal tail of the native rat protein (signal-core-tail). When examined by Edman degradation and MS, the engineered proteins were shown to have the expected N-terminal amino acid sequences and molecular masses. The native pig was found to be acetylated at the N-terminal. The native pig and core-tail enzymes were equally efficient at enhancing the side-chain cleavage activity of human CYP17 and the signal-core-tail was 55% as efficient. The core and signal-core constructs were completely inactive in the aforementioned reaction. All the five derivatives were reduced to varying degrees by NADPH:cytochrome P-450 (NADPH-P450) reductase and the relative efficiencies of this reduction were reminiscent of the behaviour of these derivatives in supporting the side-chain cleavage reaction. In the side-chain cleavage assay, however, NADPH-P450 reductase was used in large excess so that the reduction of cytochrome b5 derivatives was not rate-limiting. The results highlight that productive interaction between cytochrome b5 and CYP17 is governed not only by the presence of a membrane insertable hydrophobic region on the cytochrome b5 but also by its defined spatial orientation at the C-terminal.

Acetaldehyde↗
PubMed1997

Glycine-induced extracellular secretion of a recombinant cytochrome expressed in Escherichia coli.

The effect of each of 20 different amino acid supplements to the growth medium of Escherichia coli on the extracellular release of a periplasmic recombinant cytochrome b5 was investigated. Only glycine, and to a lesser extent histidine, stimulated the synthesis of secretory cytochrome b5, as well as its discharge into the medium. Extracellular amounts of cytochrome b5 accrued with increasing concentrations of exogenous glycine and duration of the culture period, in spite of the fact that increasing glycine in the medium progressively inhibited cell growth. For example, 1% medium glycine caused a 50% reduction in bacterial growth, but doubled the periplasmic pool of cytochrome b5 to over 25 micrograms of cytochrome b5/ml of culture at 24 h, a period during which almost all of cellular haemoprotein pool was turned over into the medium. A comparative study of the exportable form of cytochrome b5 with a (non-secretory) cytoplasmic-resident counterpart indicated that the periplasmic cytochrome b5 content was selectively discharged into the medium when less than 1% glycine was present, but, at higher doses, a significant proportion of the additional extracellular haemoprotein was derived from cell lysis. Optimal level of periplasmic discharge of the cytochrome required both active protein synthesis and the presence of a glycine supplement in the medium from the onset of bacterial growth. Phase-contrast and scanning electron microsocopy of glycine-grown Escherichia coli showed that the cells had a 3-7-fold enlarged "eyeball' spheroidal morphology, with a condensed pericircular cytoplasm. The bulk of the volume in such hypertrophied cells consisted of the periplasm; this was reflected by the progressively lowered buoyancy of E. coli cultured with increasing amounts of glycine. The fragility of such cells was apparent by their marked sensitivity to lysis at glycine concentrations above 1%. We conclude that supplementation of E. coli cultures with moderate amounts of glycine substantially stimulates the synthesis of exportable proteins and further enhances their yield by discharge into the growth medium.

Animals↗
PubMed1996

Expression, isolation, and characterization of a signal sequence-appended chimeric precursor protein.

This report describes the properties and the functional utility of an unprocessed precursor protein overproduced in Escherichia coli. The precursor protein is from a fusion between DNA sequences coding for the alkaline phosphatase signal sequence and the full-length of rat liver cytochrome b(5). The intact precursor protein accumulated in the membranes represented to over 5% of the total bacterial protein. A procedure involving disruption of the bacterial cells by sonication, isolation of the membranes by differential centrifugation, solubilization with a polar solvent, and ion-exchange chromatography provided milligram quantities of the undegraded precursor in a homogeneous and soluble form. The chimeric precursor protein displayed a characteristic b-type hemoprotein spectrum, identical to that of the native cytochrome b(5). The properties of the precursor protein have been examined by a range of biophysical and biochemical methods. Molecular modeling suggests an amphipathic structure in which a fully preserved soluble core of cytochrome b(5) is terminally bonded by hydrophobic interactions between the amino-terminal signal sequence and the carboxy-terminal membrane anchoring hemoprotein sequence. The precursor substrate was recognized and efficiently cleaved by signal peptidase.

Alkaline Phosphatase↗