Henrik W. Anthonsen

Lipases and esterases: a review of their sequences, structure and evolution

This chapter aims to provide a brief review on the enzyme family of lipases and esterases. The sequences, 3D structures and pH dependent electrostatic signatures are presented and analyzed. Since the family comprises more than 100 sequences, we have tried to focus on the most interesting features from our perspective, which translates into finding similarities and differences between members of this family, in particular in and around the active sites, and to identify residues that are partially or totally conserved. Such residues we believe are either important for maintaining the structural scaf-fold of the protein or to maintain activity or specificity. The structure function relationship for these proteins is therefore of central interest. Can we uniquely identify a protein from this large family of sequences--and if so, what is the identifier? The protein family displays some highly complex features: many of the proteins are interfacially activated, i.e. they need to be in physical contact with the aggregated substrate. Access to the active site is blocked with either a loop fragment or an alpha-helical fragment in the absence of interfacial contact. Although the number of known, relevant protein 3D structures is growing steadily, we are nevertheless faced with a virtual explosion in the number of known or deduced amino acid sequences. It is therefore unrealistic to expect that all protein sequences within the foreseeable future will have their 3D structure determined by X-ray diffractional analysis or through other methods. When feasible the gene and/or the amino acid sequences will be analyzed from an evolutionary perspective. As the 3D folds are often remarkably similar, both among the triglyceride lipases as well as among the esterases, the functional diversities (e.g. specificity) must originate in differences in surface residue utilization, in particular of charged residues. The pH variations in the isopotential surfaces of some of the most interesting lipases are presented and a qualitative interpretation proposed. Finally we illustrate that NMR has potential for becoming an important tool in the study of lipases, esterases and their kinetics.

Intracellular manganese ions provide strong T1 relaxation in rat myocardium.

The efficacy of manganese ions (Mn2+) as intracellular (ic) contrast agents was assessed in rat myocardium. T1 and T2 and Mn content were measured in ventricular tissue excised from isolated perfused hearts in which a 5‐min wash‐in with 0, 30, 100, 300, or 1000 μM of Mn dipyridoxyl diphosphate (MnDPDP) was followed by a 15‐min wash‐out to remove extracellular (ec) Mn2+. An inversion recovery (IR) analysis at 20 MHz revealed two T1 components: an ic and short T1‐1 (650–251 ms), and an ec and longer T1‐2 (2712–1042 ms). Intensities were about 68% and 32%, respectively. Tissue Mn content correlated particularly well with ic R1‐1. A two‐site water‐exchange analysis of T1 data documented slow water exchange with ic and ec lifetimes of 11.3 s and 7.5 s, respectively, and no differences between apparent and intrinsic relaxation parameters. Ic relaxivity induced by Mn2+ ions in ic water was as high as 56 (s mM)−1, about 8 times and 36 times higher than with Mn2+ aqua ions and MnDPDP, respectively, in vitro. This value is as high as any reported to date for any synthetic protein‐bound metal chelate. The increased rotational correlation time (τR) between proton and electron (Mn2+) spins, and maintained inner‐sphere water access, might make ic Mn2+ ions and Mn2+‐ion‐releasing contrast media surprisingly effective for T1‐weighted imaging. Magn Reson Med 52:506–514, 2004.

Co-solvent enhancement of enantioselectivity in lipase-catalysed hydrolysis of racemic esters. A process for production of homochiral C-3 building blocks using lipase B from Candida antarctica

Abstract Lipase-catalysed hydrolysis of butanoates of 3-methoxy-1-(phenylmethoxy)-2-propanol and 3-chloro-1-(phenylmethoxy)-2-propanol with various lipases gave low enantioselectivity, E . By additon of water miscible organic cosolvents, in particular tert -butanol and acetone, the E -value was raised from 7 to 220 for the useful chloro derivative. This finding has led to proposal of a process for production of homochiral C-3 synthons such as both enantiomers of phenylmethyl glycidyl ether starting from racemic epichlorohydrin. NMR studies of lipase B from Candida antarctica show that the conformation most likely is not changed upon addition of up to 50% acetone. Nuclear Overhause effects were observed upon irradiation of the phenyl protons of the substrate only in the presence of enzyme thus indicating an interaction between the two.

Calculation of enantiomer ratio and equilibrium constants in biocatalytic ping-pong bi-bi resolutions

Abstract A computer program for the determination of kinetic and thermodynamic parameters in biocatalytic ping-pong bi-bi resolutions has been developed. The program uses enantiomeric excesses of both product ( ee p ) and remaining substrate ( ee s ) measured at more than one conversion (ξ), and determines both the equilibrium constant K eq , the enantiomer ratio E and the selectivity factor α. The program has been tested for transesterification of 1-phenoxy-2-propanol using different excesses of acyl donor.

The enantiomer ratio strongly depends on the alkyl part of the acyl donor in transesterification with lipase B from Candida antarctica

Abstract Three secondary alcohols, 1-phenoxy-, 1-phenylmethoxy- and 1-(2-phenylethoxy)-2-propanol, have been resolved by transesterification with the acyl donors 2-chloroethyl butanoate, 2,2,2-trichloroethyl butanoate, vinyl butanoate and butanoic anhydride using lipase B from Candida antarctica as catalyst in hexane. The enantiomer ratio E , which was calculated on the basis of a ping-pong bi-bi mechanism, was highest when 2-chloroethyl butanoate was used as acyl donor, however the reaction was reversible. It was shown that the E -value increased when the amount of 2,2,2-trichloroethyl butanoate was reduced. It is also indicated that butanoic anhydride and vinyl butanoate make the lipase less specific probably by acylation.

The blind watchmaker and rational protein engineering

Abstract In the present review some scientific areas of key importance for protein engineering are discussed, such as problems involved in deducting protein sequence from DNA sequence (due to posttranscriptional editing, splicing and posttranslational modifications), modelling of protein structures by homology, NMR of large proteins (including probing the molecular surface with relaxation agents), simulation of protein structures by molecular dynamics and simulation of electrostatic effects in proteins (including pH-dependent effects). It is argued that all of these areas could be of key importance in most protein engineering projects, because they give access to increased and often unique information. In the last part of the review some potential areas for future applications of protein engineering approaches are discussed, such as non-conventional media, de novo design and nanotechnology.

A putative DNA-binding domain in the NUCKS protein

We have studied the DNA-binding properties of a NUCKS-derived, synthetic peptide containing an extended GRP motif. This peptide binds to random-sequence DNA, but did not bind preferentially to poly(dA-dT). A synthetic peptide with the same amino acid composition but with a random sequence did not bind to DNA, suggesting that the structure of the DNA-binding domain plays a pivotal role in the interaction with DNA. NMR and graphic modeling were employed to investigate the structure of the synthetic peptide. It was shown that the DNA-binding peptide constituted an alpha helix in phosphate buffer at pH 5.5. Docking results indicated an almost perfect fit for this small, helical peptide into the major groove of DNA with the possibility of four basic residues interacting with the phosphate backbone of DNA. One consensus site for phosphorylation by Cdk1 is located in the N-terminal end of the DNA-binding peptide. Upon phosphorylation of this site, the binding to DNA was completely prohibited. Immunofluorescence experiments showed that NUCKS was located in the nuclei in proliferating cells in interphase of the cell cycle, but was distributed throughout the cytoplasm in mitotic cells.

Exploring the separate NMR responses from crude oil and water in rock cores

In analysis of transverse relaxation time (T2) curves in a Carr-Purcell-Meiboom-Gill (CPMG) experiment in a multicomponent system originating from measurements of oil and water in rock cores, where internal magnetic field gradients broaden the line widths significantly, there is very little direct information to be extracted of the different components contributing to the totalT2 relaxation time curve. From the study of rock cores saturated with different amounts of crude oil and water, we show that with an optimised experimental setup it is possible to extract information from the nuclear magnetic resonance response that is not resolved by any other methods. This setup combines pulsed field gradient methods with the CPMG experiment utilizing data from both rock cores and bulk oil and water. Then it becomes feasible to separate the signals from oil and water where the two-dimensional inverse Laplace transform ordinarily seems to fail.

Combining PFG and CPMG NMR measurements for separate characterization of oil and water simultaneously present in a heterogeneous system

When analyzingT2 relaxation time curves from an ordinary Carr-Purcell-Meiboom-Gill (CPMG) experiment in a multicomponent system, where internal magnetic field gradients broaden the line widths significantly, there is very little direct information regarding the mobility of the components and on the type of environment experienced by each component. Compared to a standard CPMG experiment, a combination of pulsed field gradient (PFG) methods with the CPMG experiment will increase the amount of information that is obtainable from the nuclear magnetic resonance (NMR) experiment on a system of components differing significantly in molecular mobility. We propose a method for achieving separate measurements of theT2 attenuation of two components simultaneously present within a sample, and we believe it to be generally valid for any system in which the components differ significantly in molecular mobility. The two components could be oil and water in porous rock, or fat and water in a biological tissue, where a separation of theT2 attenuations for the two components will add insight to the study of the systems. In order to verify the method we made use of a sample containing a mixture of oil and water in two separate bulk phases, and compared the results with PFG-CPMG experiments performed on samples containing oil or water only, respectively. The method was applied to systems containing glass spheres immersed in water and oil, and it was possible to obtain information about the physical environment of the components which otherwise is not easily obtainable. The method presented here is therefore presumably applicable to whole rock cores or tissue samples.

Manganese-calcium interactions with contrast media for cardiac magnetic resonance imaging: a study of manganese chloride supplemented with calcium gluconate in isolated Guinea pig hearts.

Objectives:Manganese ions (Mn2+) enter cardiomyocytes via calcium (Ca2+) channels and enhance relaxation intracellularly. To prevent negative inotropy, new Mn2+-releasing contrast agents have been supplemented with high Ca2+. The study aim was to investigate how this affects cardiac function and magnetic resonance efficacy. Materials and Methods:MnCl2 based contrast agents, manganese and manganese–calcium (Ca2+:Mn2+ 10:1), were infused during 4 repeated washin–washout sequences in perfused guinea pig hearts. [Mn2+] were 10, 50, 100 and 500 μM. Results:During washin, manganese depressed left ventricular developed pressure (LVDP) by 4, 9, 17, and 53% whereas manganese–calcium increased LVDP by 13, 18, 25, and 56%. After experiments, tissue Mn contents (nmol/g dry wt) were control <40, manganese 3720, and manganese–calcium 1620. T1 was reduced by 85–92% in Mn2+-enriched hearts. Conclusions:High Ca2+ supplements to Mn2+-releasing contrast agents may be counterproductive by inducing a strong positive inotropic response and by reducing the magnetic resonance efficacy.