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Objectives: Fetal mutations and fetal chromosomal abnormalities can be detected by molecular analysis of circulating cell free fetal DNA (ccffDNA) from maternal plasma. This comprehensive study was aimed to investigate and verify blood collection and blood shipping conditions that enable Noninvasive Prenatal Testing. Specifically, the impact of shipping and storage on the stability and concentration of circulating cell-free DNA (ccfDNA) in Streck® Cell-Free DNA™ Blood Collection Tubes (Streck BCTs, Streck, Omaha NE). These BCTs were designed to minimize cellular degradation, and thus effectively prevent dilution of fetal ccf DNA by maternal genomic DNA, was evaluated., Design and Methods: Peripheral venous maternal blood was collected into Streck BCTs to investigate four aspects of handling and processing conditions: (1) time from blood draw to plasma processing; (2) storage temperature; (3) mechanical stress; and (4) lot-to-lot tube variations., Results: Maternal blood stored in Streck BCTs for up to 7 days at ambient temperature provides stable concentrations of ccffDNA. The amount of fetal DNA did not change over a broad range of storage temperatures (4°C, 23°C, 37°C, 40°C), but the amount of total (largely maternal) DNA increased in samples stored at 23°C and above, indicating maternal cell degradation and genomic DNA release at elevated temperatures. Shipping maternal blood in Streck BCTs, did not affect sample quality., Conclusions: Maternal plasma DNA stabilized for 0 to 7 days in Streck BCTs can be used for non-invasive prenatal molecular applications, when temperatures are maintained within the broad parameters assessed in this study., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

2-Deoxyribose-5-phosphate aldolase (DERA, EC 4.1.2.4) catalyzes the reversible aldol reaction between acetaldehyde and D-glyceraldehyde-3-phosphate to generate D-2-deoxyribose-5-phosphate. It is unique among the aldolases as it catalyzes the reversible asymmetric aldol addition reaction of two aldehydes. In order to expand the substrate scope and stereoselectivity of DERA, structure-based substrate design as well as site-specific mutation has been investigated. Using the 1.05 A crystal structure of DERA in complex with its natural substrate as a guide, five site-directed mutants were designed in order to improve its activity with the unnatural nonphosphorylated substrate, D-2-deoxyribose. Of these, the S238D variant exhibited a 2.5-fold improvement over the wild-type enzyme in the retroaldol reaction of 2-deoxyribose. Interestingly, this S238D mutant enzyme was shown to accept 3-azidopropinaldehyde as a substrate in a sequential asymmetric aldol reaction to form a deoxy-azidoethyl pyranose, which is a precursor to the corresponding lactone and the cholesterol-lowering agent Lipitor. This azidoaldehyde is not a substrate for the wild-type enzyme. Another structure-based design of new nonphosphorylated substrates was focused on the aldol reaction with inversion in enantioselectivity using the wild type or the S238D variant as the catalyst and 2-methyl-substituted aldehydes as substrates. An example was demonstrated in the asymmetric synthesis of a deoxypyranose as a new effective synthon for the total synthesis of epothilones. In addition, to facilitate the discovery of new enzymatic reactions, the engineered E. coli strain SELECT (Deltaace, adhC, DE3) was developed to be used in the future for selection of DERA variants with novel nonphosphorylated acceptor specificity.

A transition state analogue inhibitor, boronic acid benzophenone (BBP) photoprobe, was used to study the differences in the topology of the S1 pocket of chemically modified mutant enzymes (CMMs). The BBP proved to be an effective competitive inhibitor and a revealing active site directed photoprobe of the CMMs of the serine protease subtilisin Bacillus lentus (SBL) which were chemically modified with the hydrophobic, negatively charged and positively charged moieties at the S1 pocket S166C residue. As expected, in all cases BBP bound best to WT-SBL. BBP binding to S166C-SCH2C6H5 and S166C-CH2-c-C6H11, with their large hydrophobic side chains, was reduced by 86-fold and 9-fold, respectively, compared to WT. Relative to WT, BBP binding to the charged CMMs, S166C-S-CH2CH2SO3- or S166C-S-CH2CH2NH3+, was reduced 170-fold and 4-fold respectively. Photolysis of the WT-SBL-BBP enzyme inhibitor (EI) complex, inactivated the enzyme and effected the formation of a covalent crosslink between WT and BBP. The crosslink was identified at Gly127 by peptide mapping analysis and Edman sequencing. Gly127 is located in the S1 hydrophobic pocket of SBL and its modification thus established binding of the benzophenone moiety in S1. Photolysis of the EI complex of S166C-SCH2C6H5, S166C-S-CH2CH2SO3-, or S166C-S-CH2CH2NH3+ and BBP under the same conditions did not inactivate these enzymes, nor effect the formation of a crosslink. These results corroborated the kinetic evidence that the active site topology of these CMMs is dramatically altered from that of WT. In contrast, while photolysis of the S166C-CH2-c-C6H11-BBP EI complex only inactivated 50% of the enzyme after 12 h, it still effected the formation of a covalent crosslink between the CMM and BBP, again at Gly127. However, this photolytic reaction was less efficient than with WT, demonstrating that the S1 pocket of S166C-CH2-c-C6H11 is significantly restricted compared to WT, but not as completely as for the other CMMs.

The use of methanethiosulfonates as thiol-specific modifying reagents in the strategy of combined site-directed mutagenesis and chemical modification allows virtually unlimited opportunities for creating new protein surface environments. As a consequence of our interest in electrostatic manipulation as a means of tailoring enzyme activity and specificity, we have adopted this approach for the controlled incorporation of multiple negative charges at single sites in the representative serine protease, subtilisin Bacillus lentus (SBL). A series of mono-, di- and triacidic acid methanethiosulfonates were synthesized and used to modify cysteine mutants of SBL at positions 62 in the S2 site, 156 and 166 in the S1 site and 217 in the S1' site. Kinetic parameters for these chemically modified mutant (CMM) enzymes were determined at pH 8.6 under conditions which ensured complete ionization of the unnatural amino acid side-chains introduced. The presence of up to three negative charges in the S1, S1' and S2 subsites of SBL resulted in up to 11-fold lowered activity, possibly due to interference with oxyanion stabilization of the transition state of the hydrolytic reactions catalyzed. Each unit increase in negative charge resulted in a raising of K(M) and a reduction of k(cat). However, no upper limit was observed for increases in K(M), whereas decreases in k(cat) reached a limiting value. Comparison with sterically similar but uncharged CMMs revealed that electrostatic effects of negative charges at positions 62, 156 and 217 are detrimental, but are beneficial at position 166. These results indicate that the ground-state binding of SBL to the standard substrate, Suc-AAPF-pNA, to SBL is reduced, but without drastic attenuation of catalytic efficiency, and show that SBL tolerates high levels of charge at single sites.

A series of chemically modified mutants (CMMs) of subtilisin B. lentus (SBL) were generated employing the combination of site-directed mutagenesis and chemical modification. This strategy entails the mutation of a selected active site residue to cysteine and its subsequent modification with a methanethiosulfonate reagent CH3SO2S-R, where R may be infinitely variable. The present study was undertaken to evaluate the changes in specificity and pH-activity profiles that could be induced by modification of S156C and S166C in the S1 pocket of SBL with a representative range of side chain modifications, namely R=-CH3, -CH2C6H5, -CH2CH2NH3+ and CH2CH2SO3 . The side chain of S156C is surface exposed and well solvated while that of S166C points into the pocket. Kinetic evaluation of the CMMs with suc-AAPF-pNA as substrate showed that the kcat/K(M)s changed very little for the S156C CMMs, but varied by up to 11-fold for the S166C CMMs. pH-Activity profiles were also determined, and showed that a negatively or positively charged side chain modification increased or decreased respectively, the pKa of the catalytic triad histidine for both modification sites but with more dramatic changes for the interior pointing S166C than for the solvent exposed S156C site. As an additional probe of altered specificity, inhibition of the CMMs by a representative series of 5 boronic acid transition state analogue inhibitors was determined. The K(I)s observed ranged from a 3.5-fold improvement over the WT value, to a 12-fold decrease in binding. Overall, greater variability in all the parameters measured, activity, pKa, and boronic acid binding resulted from modification at the inward pointing 166 site than at the solvent-exposed 156 site.