Alanine resolution

A hollow-fiber liquid membrane was used in a separation of D,L-lactic acid and D,L-alanine resolution [196]. In this case, the enantioselective transport of solutes performed in one module was facihtated by N-3,5-dinitrobenzoyl-L-alanine octylester chiral selector, dissolved in toluene. The maximum D,L-lactic acid separation factor achieved was 2.00 and that for the D,L-alanine was 1.75. In both cases, the D-enantiomer flux was preferred. These values correspond to the enantiomeric excess 33.5% ee and 27.2% ee, respectively, and are not as good as in the first example. However, note that in this case, only one separation step took place and feed phase was circulated in the module. 

From a map at low resolution (5 A or higher) one can obtain the shape of the molecule and sometimes identify a-helical regions as rods of electron density. At medium resolution (around 3 A) it is usually possible to trace the path of the polypeptide chain and to fit a known amino acid sequence into the map. At this resolution it should be possible to distinguish the density of an alanine side chain from that of a leucine, whereas at 4 A resolution there is little side chain detail. Gross features of functionally important aspects of a structure usually can be deduced at 3 A resolution, including the identification of active-site residues. At 2 A resolution details are sufficiently well resolved in the map to decide between a leucine and an isoleucine side chain, and at 1 A resolution one sees atoms as discrete balls of density. However, the structures of only a few small proteins have been determined to such high resolution. 

The improvements in resolution achieved in each deconvolution step are shown in Figure 3-3. While the initial library could only afford a modest separation of DNB-glutamic acid, the library with proline in position 4 also separated DNP derivatives of alanine and aspartic acid, and further improvement in both resolution and the number of separable racemates was observed for peptides with hydrophobic amino acid residues in position 3. However, the most dramatic improvement and best selectivity were found for c(Arg-Lys-Tyr-Pro-Tyr-(3-Ala) (Scheme 3-2a) with the tyrosine residue at position 5 with a resolution factor as high as 28 observed for the separation of DNP-glutamic acid enantiomers. 

The phosphotriesterase from Pseudomonas diminuta was shown to catalyze the enantioselective hydrolysis of several racemic phosphates (21), the Sp isomer reacting faster than the Rp compound [65,66]. Further improvements using directed evolution were achieved by first carrying out a restricted alanine-scan [67] (i.e. at predetermined amino acid positions alanine was introduced). Whenever an effect on activity/ enantioselectivity was observed, the position was defined as a hot spot. Subsequently, randomization at several hot spots was performed, which led to the identification of several highly (S)- or (R)-selective mutants [66]. A similar procedure was applied to the generation of mutant phosphotriesterases as catalysts in the kinetic resolution of racemic phosphonates [68]. 

A high-resolution 1 1 solution NMR structure of lmPy-y9-lm-y9-lmPy-y9-Dp elucidated the role of /9-alanine in minor groove recognition (Fig. 3.7 b) [47]. The p residues allow both Im rings in the /9-lm-/9-lm subunit to adapt to the relatively large... 

Fumaric acid to L-aspartic acid, L-aspartic acid to L-alanine Enzymatic resolution of methyl ester of (+/-) trans 4- methoxy-... 

Figure 1. Amplitudes of the Fourier coefficients of log(model density, from a multipolar tit to 23 K diffraction data protect [45]. Continuous line m(x) = uniform distribution. Dotted line m(x) = core and valence monopoles. The vertical bar marks the experimental resolution limit 0.463 A. 

Figure 1 shows the average strength of the Fourier coefficients of log( (x)/m(x)), with q(x) a multipolar synthetic density for L-alanine at 23 K, and two different prior-prejudice distributions mix). It is apparent that the exponential needed to modulate the uniform prior still has Fourier coefficients larger than 0.01 past the experimental resolution limit of 0.463 A. Any attempt at fitting the corresponding experimental structure factor amplitudes by modulation of the uniform prior-prejudice distribution will therefore create series termination ripples in the resulting MaxEnt distribution. 

To check this prediction, a number of MaxEnt charge density calculations have been performed with the computer program BUSTER [42] on a set of synthetic structure factors, obtained from a reference model density for a crystal of L-alanine at 23 K. The set of 1500 synthetic structure factors, complete up to a resolution of 0.555 A [45], was calculated from a multipolar expansion of the density, with the computer program VALRAY[ 46],... 

BUSTER chooses the minimal grid necessary to avoid aliasing effects, based on the prior prejudice used and on the fall-off of the structure factor amplitudes with resolution for the 23 K L-alanine valence density reconstruction the grid was (64 144 64). The cell parameters for the crystal are a = 5.928(1)A b = 12.260(2)A c = 5.794(1) A [45], so that the grid step was shorter than 0.095 A along each axis. 

We briefly discuss in this section the results of the valence MaxEnt calculation on the noisy data set for L-alanine at 23 K we will denote this calculation with the letter A. The distribution of residuals at the end of the calculation is shown in Figure 5. It is apparent that no gross outliers are present, the calculated structure factor amplitudes being within 5 esd s from the observed values at all resolution ranges. 

Fig. 18. The active site region of the electron density difference map between N-carbobenzoxy-L-alanine-elastase at —SS C and native elastase at the same temperature. The resolution is 3.5 A. The bilobed feature is consistent with the binding of the alanyi portion of the substrate to the oxygen of the catalytic serine, with weak interaction of the carbobenzoxy group to the surface of the enzyme.

LeMagueres, R Im, H. Dvorak, A. Strych, U. Benedik, M. Krause, K. L. Crystal Structure at 1.45 A Resolution of Alanine Racemase from a Pathogenic Bacterium, Pseudomonas aeruginosa, Contains Both Internal and External Aldimine Forms. Biochemistry 2003, 42, 14752-14761. 

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