Biomedical subjects

F Hannemann

Publications and source records attributed to F Hannemann.

7 recordsLinked to original sources

PubMed2006

Function and engineering of the 15beta-hydroxylase CYP106A2.

CYP106A2 from Bacillus megaterium ATCC 13368 is a bacterial cytochrome P450 that is capable of transforming steroid hormones. It can be easily expressed in Escherichia coli with a high yield. Its activity in vitro can be achieved by using the adrenal redox proteins adrenodoxin and adrenodoxin reductase. So far, it was not possible to crystallize CYP106A2 because of degradation during the crystallization process. Nevertheless, CYP106A2 is an interesting enzyme for biotechnological use. It hydroxylates pharmaceutically important steroids such as progesterone and 11-deoxycortisol. However, it will be necessary for efficient application of CYP106A2 in biotechnology to improve the hydroxylation activity and manipulate the regiospecificity. The present paper gives an overview of recent developments in protein engineering of CYP106A2.

Bacillus megaterium↗

PubMed2001

Functional display of active bovine adrenodoxin on the surface of E. coli by chemical incorporation of the [2Fe-2S] cluster.

The display of heterologous proteins on the surface of living cells bears promising options for a wide variety of biotechnological applications. Up to now, however, cellular surface display was merely restricted to simple polypeptide chains. Here we present for the first time the efficient display of a protein (bovine adrenodoxin) that contains an inorganic, prosthetic group in its active form on the surface of Escherichia coli. For this purpose apo-adrenodoxin was transported to the cell surface and anchored within the outer membrane by the autotransporter pathway. Incorporation of the iron-sulfur cluster was achieved by a single-vial, one-step titration under anaerobic conditions. The biological function of surface-displayed holo-adrenodoxin could be established through adrenodoxin-dependent steroid conversion by two different cytochrome P450 enzymes and the number of functional molecules on the cell surface could be determined to be more than 10(5) per cell. Neither the expression of adrenodoxin nor the incorporation of the chemical iron-sulfur cluster reduced the viability of the bacterial cells.

Adrenodoxin↗

PubMed2001

The interaction of bovine adrenodoxin with CYP11A1 (cytochrome P450scc) and CYP11B1 (cytochrome P45011beta ). Acceleration of reduction and substrate conversion by site-directed mutagenesis of adrenodoxin.

The kinetics of protein-protein interaction and heme reduction between adrenodoxin wild type as well as eight mutants and the cytochromes P450 CYP11A1 and CYP11B1 was studied in detail. Rate constants for the formation of the reduced CYP11A1.CO and CYP11B1.CO complexes by wild type adrenodoxin, the adrenodoxin mutants Adx-(4-108), Adx-(4-114), T54S, T54A, and S112W, and the double mutants Y82F/S112W, Y82L/S112W, and Y82S/S112W (the last four mutants are Delta113-128) are presented. The rate constants observed differ by a factor of up to 10 among the respective adrenodoxin mutants for CYP11A1 but not for CYP11B1. According to their apparent rate constants for CYP11A1, the adrenodoxin mutants can be grouped into a slow (wild type, T54A, and T54S) and a fast group (all the other mutants). The adrenodoxin mutants forming the most stable complexes with CYP11A1 show the fastest rates of reduction and the highest rate constants for cholesterol to pregnenolone conversion. This strong correlation suggests that C-terminal truncation of adrenodoxin in combination with the introduction of a C-terminal tryptophan residue enables a modified protein-protein interaction rendering the system almost as effective as the bacterial putidaredoxin/CYP101 system. Such a variation of the adrenodoxin structure resulted in a mutant protein (S112W) showing a 100-fold increased efficiency in conversion of cholesterol to pregnenolone.

Adrenodoxin↗

PubMed2001

The loop region covering the iron-sulfur cluster in bovine adrenodoxin comprises a new interaction site for redox partners.

The amino acid in position 49 in bovine adrenodoxin is conserved among vertebrate [2Fe-2S] ferredoxins as hydroxyl function. A corresponding residue is missing in the cluster-coordinating loop of plant-type [2Fe-2S] ferredoxins. To probe the function of Thr-49 in a vertebrate ferredoxin, replacement mutants T49A, T49S, T49L, and T49Y, and a deletion mutant, T49Delta, were generated and expressed in Escherichia coli. CD spectra of purified proteins indicate changes of the [2Fe-2S] center geometry only for mutant T49Delta, whereas NMR studies reveal no transduction of structural changes to the interaction domain. The redox potential of T49Delta (-370 mV) is lowered by approximately 100 mV compared with wild type adrenodoxin and reaches the potential range of plant-type ferredoxins (-305 to -455 mV). Substitution mutants show moderate changes in the binding affinity to the redox partners. In contrast, the binding affinity of T49Delta to adrenodoxin reductase and cytochrome P-450 11A1 (CYP11A1) is dramatically reduced. These results led to the conclusion that Thr-49 modulates the redox potential in adrenodoxin and that the cluster-binding loop around Thr-49 represents a new interaction region with the redox partners adrenodoxin reductase and CYP11A1. In addition, variations of the apparent rate constants of all mutants for CYP11A1 reduction indicate the participation of residue 49 in the electron transfer pathway between adrenodoxin and CYP11A1.

Adrenodoxin↗

PubMed2001

Cloning and expression analysis of NhL1, a gene encoding an extracellular lipase from the fungal pea pathogen Nectria haematococca MP VI (Fusarium solani f. sp. pisi) that is expressed in planta.

The filamentous fungus Nectria haematococca (anamorph Fusarium solani f. sp. pisi) resides in soil, and attacks pea seedlings in the area of the underground epicotyl and upper tap root, causing foot rot disease. We detected lipase activity during in vitro growth of N. haematococca. Subsequently, a lipase gene was cloned and functionally characterised by heterologous expression in Saccharomyces cerevisiae. The full-length cDNA of 1152 bp was cloned using a 3' RACE-PCR approach coupled with cDNA library screening. The genomic clone, comprising an ORF of 999 bp interrupted by two introns of 56 and 64 bp, was isolated from a newly constructed lambda phage library. Analysis of the deduced protein sequence revealed the presence of a typical signal peptide at the N-terminus, and of the three conserved amino acids forming the active site of lipases. The lipase of N. haematococca has a low degree of similarity to the lipases from Humicola lanuginosa (37.2%), Rhizomucor miehei (21.6%), Rhizopus delemar (23.1%), Rhizopus niveus (25.9%), and to mono- and diacylglycerol lipase from Penicillium camembertii (30.8%), and very high similarity (94.6%) to a lipase from Fusarium heterosporum. The lipase from N. haematococca shows maximal activity at 37 degrees C and pH 8.0. Based on Southern analysis, the lipase clone represents a single-copy gene in N. haematococca. Expression analysis was performed by RT-PCR. In vitro, the lipase gene shows a low basal expression, but is highly inducible by lipase substrates, and repressed by glucose. During plant infection, transcripts of this fungal lipase gene were detected 4, 8, and 10 days after infection.

Amino Acid Sequence↗

PubMed2000

Adrenodoxin: structure, stability, and electron transfer properties.

Adrenodoxin is an iron-sulfur protein that belongs to the broad family of the [2Fe-2S]-type ferredoxins found in plants, animals and bacteria. Its primary function as a soluble electron carrier between the NADPH-dependent adrenodoxin reductase and several cytochromes P450 makes it an irreplaceable component of the steroid hormones biosynthesis in the adrenal mitochondria of vertebrates. This review intends to summarize current knowledge about structure, function, and biochemical behavior of this electron transferring protein. We discuss the recently solved first crystal structure of the vertebrate-type ferredoxin, the truncated adrenodoxin Adx(4-108), that offers the unique opportunity for better understanding of the structure-function relationships and stabilization of this protein, as well as of the molecular architecture of [2Fe-2S] ferredoxins in general. The aim of this review is also to discuss molecular requirements for the formation of the electron transfer complex. Essential comparison between bacterial putidaredoxin and mammalian adrenodoxin will be provided. These proteins have similar tertiary structure, but show remarkable specificity for interactions only with their own cognate cytochrome P450. The discussion will be largely centered on the protein-protein recognition and kinetics of adrenodoxin dependent reactions.

Adrenodoxin↗

PubMed1991

Genetic engineering and overexpression of ribosomal L12 protein genes from three different archaebacteria in E coli.

Genes coding for ribosomal protein L12 from Methanococcus vannielii (Mva), Halobacterium halobium (Hha) and Sulfolobus solfataricus (Sso) have been subcloned in the polylinker region of pUC19. An efficient Shine-Dalgarno sequence has been attached to the 5' end of the genes, and two ochre stop codons have been created at their 3' ends, where necessary. In addition, mutants of the MvaL12 and HhaL12 genes were constructed, which coded for a cysteine residue at the C-terminus of the protein. The constructs were transferred together with the pUC19 polylinker as gene cartridges into different expression vectors. These constructed plasmids were transformed in the appropriate E coli hosts and tested for expression. Two systems were found to work efficiently for overexpression, namely the pKK223-3 vector featuring a tac promoter, and the pT7-5 vector featuring a T7-promoter. The over-expressed proteins were purified to homogeneity; their purity was investigated by one and two-dimensional gel systems, amino acid analysis and N-terminal protein sequencing for 10 steps or more. The amount of protein purified from E coli test cultures bearing the expression plasmids was always more than 2.5 mg/l of medium used.

Amino Acids↗