Production of epoxide hydrolases in batch fermentations of <Emphasis Type="Italic">Botryosphaeria rhodina</Emphasis>

The filamentous fungus Botryosphaeria rhodina (ATCC 9055) was investigated related to its ability for epoxide hydrolase (EH) production. Epoxide hydrolase activity is located at two different sites of the cells. The larger part is present in the cytosol (70%), while the smaller part is associated to membranes (30%). In media optimization experiments, an activity of 3.5 U/gDW for aromatic epoxide hydrolysis of para-nitro-styrene oxide (pNSO) could be obtained. Activity increased by 30% when pNSO was added to the culture during exponential growth. An increase of enzyme activity up to 6 U/gDW was achieved during batch-fermentations in a bioreactor with 2.7 l working volume. Evaluation of fermentations with 30 l working volume revealed a relation of oxygen uptake rate to EH expression. Oxygen limitation resulted in a decreased EH activity. Parameter estimation by the linearization method of Hanes yielded Km values of 2.54 and 1.00 mM for the substrates S-pNSO and R-pNSO, respectively. vmax was 3.4 times higher when using R-pNSO. A protein purification strategy leading to a 47-fold increase in specific activity (940 U/mgProtein) was developed as a first step to investigate molecular and structural characteristics of the EH.     

Metabolism of 2-chloro-4-methylphenoxyacetate by Alcaligenes eutrophus JMP 134

2-Chloro-4-methylphenoxyacetate is not a growth substrate for Alcaligenes eutrophus JMP 134 and JMP 1341. It is, however, being transformed by enzymes of 2,4-dichlorophenoxyacetic acid metabolism to 2-chloro-4-methyl-cis, cis-muconate, which is converted by enzymatic 1,4-cycloisomerization to 4-carboxymethyl-2-chloro-4-methylmuconolactone as a dead end metabolite. Chemically, only 3,6-cycloisomerization occurs, giving rise to both diastereomers of 4-carboxychloromethyl-3-methylbut-2-en-4-olide. Those lactones harbonring a chlorosubstituent on the 4-carboxymethyl side chain were surprisingly stable under physiological as well as acidic conditions.     

Association of the chaperone alphaB-crystallin with titin in heart muscle

alphaB-crystallin, a major component of the vertebrate lens, is a chaperone belonging to the family of small heat shock proteins. These proteins form oligomers that bind to partially unfolded substrates and prevent denaturation. alphaB-crystallin in cardiac muscle binds to myofibrils under conditions of ischemia, and previous work has shown that the protein binds to titin in the I-band of cardiac fibers (Golenhofen, N., Arbeiter, A., Koob, R., and Drenckhahn, D. (2002) J. Mol. Cell. Cardiol. 34, 309-319). This part of titin extends as muscles are stretched and is made up of immunoglobulin-like modules and two extensible regions (N2B and PEVK) that have no well defined secondary structure. We have followed the position of alphaB-crystallin in stretched cardiac fibers relative to a known part of the titin sequence. alphaB-crystallin bound to a discrete region of the I-band that moved away from the Z-disc as sarcomeres were extended. In the physiological range of sarcomere lengths, alphaB-crystallin bound in the position of the N2B region of titin, but not to PEVK. In overstretched myofibrils, it was also in the Ig region between N2B and the Z-disc. Binding between alphaB-crystallin and N2B was confirmed using recombinant titin fragments. The Ig domains in an eight-domain fragment were stabilized by alphaB-crystallin; atomic force microscopy showed that higher stretching forces were needed to unfold the domains in the presence of the chaperone. Reversible association with alphaB-crystallin would protect I-band titin from stress liable to cause domain unfolding until conditions are favorable for refolding to the native state.     

Exploring routes to stabilize a cationic pyridoxamine in an artificial transaminase: site-directed mutagenesis versus synthetic cofactors

Two artificial transaminases were assembled by linking a pyridoxamine derivative within an engineered fatty acid binding protein. The goal of mimicking a native transamination site by stabilizing a cationic pyridoxamine ring system was approached using two different strategies. First, the scaffold of intestinal fatty acid binding protein (IFABP) was tailored by molecular modeling and site-directed mutagenesis to position a carboxylate group close to the pyridine nitrogen of the cofactor. When these IFABP mutants (IFABP-V60C/L38K/E93E and -V60C/E51K/E93E) proved to be unstable, a second approach was explored. By N-methylation of the pyridoxamine, a cationic cofactor was created and tethered to Cys60 of IFABP-V60C/L38K and -V60C/E51K; this latter strategy had the effect of permanently installing a positive charge on the cofactor. These chemogenetic assemblies catalyze the transamination between alpha-ketoglutarate and various amino acids with enantioselectivities of up to 96% ee. The pH profile of the initial rates is bell shaped and similar to native aminotransferases. The k(cat) values and the turnover numbers for these new constructs are the highest achieved to date in our system. This success was only made possible by the unique flexibility of the underlying enzyme design concept employed, which permits full control of both the protein scaffold and the catalytically active group.     

Electron transfer in cyanobacterial photosystem I: II. Determination of forward electron transfer rates of site-directed mutants in a putative electron transfer pathway from A0 through A1 to FX

The directionality of electron transfer in Photosystem I (PS I) is investigated using site-directed mutations in the phylloquinone (QK) and FX binding regions of Synnechocystis sp. PCC 6803. The kinetics of forward electron transfer from the secondary acceptor A1 (phylloquinone) were measured in mutants using time-resolved optical difference spectroscopy and transient EPR spectroscopy. In whole cells and PS I complexes of the wild-type both techniques reveal a major, slow kinetic component of tau approximately 300 ns while optical data resolve an additional minor kinetic component of tau approximately 10 ns. Whole cells and PS I complexes from the W697FPsaA and S692CPsaA mutants show a significant slowing of the slow kinetic component, whereas the W677FPsaB and S672CPsaB mutants show a less significant slowing of the fast kinetic component. Transient EPR measurements at 260 K show that the slow phase is approximately 3 times slower than at room temperature. Simulations of the early time behavior of the spin polarization pattern of P700+A1-, in which the decay rate of the pattern is assumed to be negligibly small, reproduce the observed EPR spectra at 260 K during the first 100 ns following laser excitation. Thus any spin polarization from P700+FX- in this time window is very weak. From this it is concluded that the relative amplitude of the fast phase is negligible at 260 K or its rate is much less temperature-dependent than that of the slow component. Together, the results demonstrate that the slow kinetic phase results from electron transfer from QK-A to FX and that this accounts for at least 70% of the electrons. Although the assignment of the fast kinetic phase remains uncertain, it is not strongly temperature dependent and it represents a minor fraction of the electrons being transferred. All of the results point toward asymmetry in electron transfer, and indicate that forward transfer in cyanobacterial PS I is predominantly along the PsaA branch.     

Ab initio Models for Six-Center Multiple Proton Exchange and Ion Pair Formation Assisted by Lewis Acids

High level ab initio and density functional calculations, extrapolated to QCISD(T)/6-311+G(3df,2p)//MP2/6-31+G^**+ZPE, reveal that cyclic ion pairs can form in the hydrogen bonded complexes of haloboric acids BH_nX_3-n–HX, X=F, Cl, with Lewis bases HX, H₂O, CH₃OH, and NH₃, even in isolation (e.g., in the gas phase). The intrinsic acidities (deprotonation energies) required for protonation of these bases with formation of gas phase ion pairs are calculated to be <295 kcal/mol for water, <301 kcal/mol for methanol, and <306 kcal/mol for ammonia; such values are common for acidic sites in zeolites. All gas phase ion pairs prefer symmetric bidentate or tridentate structures. In the other cases where hydrogen bonded complexes prevail, symmetric ion pair-like transition structures for multiple hydrogen exchange are computed.Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s0089400060563     

Modeling the spring blooms of ciliates in a deep lake

In contrast to the macro/mesozooplankton, microzooplankton has received much less attention in ecosystem models. In many modeling studies, microzooplankton has been either entirely neglected, or else, data were often not available for validation, or agreement between the observed and the simulated abundances was rather poor. In this study, we compare the simulation results from several alternative models considering different formulations of ciliate growth in a hydrodynamically driven 1D nutrient-phytoplankton–multiple zooplankton model, with long-term datasets from the deep, monomictic Lake Constance. We show that the parameterization of the limitation of ciliate growth with a constant specific mortality rate and/or predation by copepods leads to uncontrolled ciliate blooms. In contrast, implementation of a density-dependent mortality rate enables reproduction of algae–ciliate dynamics over a variety of environmental settings encompassed by the 14-year dataset spanning 21 years in a lake undergoing oligotrophication. Considering the numerous processes that can be responsible for the dampening of ciliate blooms, our findings suggest that employing a simple density-dependent mortality term offers a pragmatic solution for the challenge of including the microzooplankton, characterized by an overwhelming complexity of trophic interactions, in ecosystem models.     

Distinct Functional Interactions between Actin Isoforms and Nonsarcomeric Myosins

Despite their near sequence identity, actin isoforms cannot completely replace each other in vivo and show marked differences in their tissue-specific and subcellular localization. Little is known about isoform-specific differences in their interactions with myosin motors and other actin-binding proteins. Mammalian cytoplasmic β- and γ-actin interact with nonsarcomeric conventional myosins such as the members of the nonmuscle myosin-2 family and myosin-7A. These interactions support a wide range of cellular processes including cytokinesis, maintenance of cell polarity, cell adhesion, migration, and mechano-electrical transduction. To elucidate differences in the ability of isoactins to bind and stimulate the enzymatic activity of individual myosin isoforms, we characterized the interactions of human skeletal muscle α-actin, cytoplasmic β-actin, and cytoplasmic γ-actin with human myosin-7A and nonmuscle myosins-2A, -2B and -2C1. In the case of nonmuscle myosins-2A and -2B, the interaction with either cytoplasmic actin isoform results in 4-fold greater stimulation of myosin ATPase activity than was observed in the presence of α-skeletal muscle actin. Nonmuscle myosin-2C1 is most potently activated by β-actin and myosin-7A by γ-actin. Our results indicate that β- and γ-actin isoforms contribute to the modulation of nonmuscle myosin-2 and myosin-7A activity and thereby to the spatial and temporal regulation of cytoskeletal dynamics. FRET-based analyses show efficient copolymerization abilities for the actin isoforms in vitro. Experiments with hybrid actin filaments show that the extent of actomyosin coupling efficiency can be regulated by the isoform composition of actin filaments.