Ryszard Michalczyk

ROTATIONAL DYNAMICS OF ADENINE AMINO GROUPS IN A DNA DOUBLE HELIX

Exocyclic amino groups of the bases undergo conformational fluctuations that affect the recognition and reactivity of nucleic acid molecules. Among these fluctuations, rotation of amino groups around C-N bonds is of special interest. In the present paper, we report the first determination of the rates and energetic parameters for rotation of the N6-amino group of adenine in a DNA double helix. The DNA molecule studied is the dodecamer [d(CGCGAGCTCGCG)]2. The adenine in each A. T basepair of the dodecamer was labeled with 15N at the N6 position, and the NMR resonances of the two protons in the adenine amino group were selectively observed by 15N-editing methods. The rates of rotation of the amino group were obtained from experiments of transfer of magnetization between the two protons in the same group and from lineshape analysis of 15N-edited amino proton NMR resonances. The results show that, over the temperature range from 0 to 70 degrees C, the rates of rotation of adenine amino groups range from 60 to 24,000 s-1. Formation of the activated state during rotation has a standard enthalpy change of 15.3 +/- 0.2 kcal/mol and a standard entropy change of 6.0 +/- 0.7 cal/(mol. K). Analysis of the results suggests that rotation of the amino group occurs in the paired, closed state of the adenine in the A. T basepair of the double-helical DNA structure.

Heteronuclear 15N and 1H Magnetic Resonance Study of a DNA Dodecamer Containing an A3T3 Tract

Two related DNA dodecamers, [d(CGCAAATTTGCG)]2 and [d(CGCGAGCTCGCG)]2, with all adenine residues 15N‐labeled at the amino groups were synthesized. The dynamics and conformational transitions in both dodecamers were investigated using 15N and 1H NMR spectroscopy. It was found that, in the premelting temperature range, the 15N resonances of the first and second adenines in the A3T3 tract of [d(CGCAAATTTGCG)]2 shift downfield with increase in temperature. The change in the 15N chemical shift of the central adenine in the A3T3 tract was an order of magnitude smaller and was similar to that of the single adenine in [d(CGCGAGCTCGCG)]2. These premelting 15N chemical shift changes parallel the pattern of three‐centered hydrogen bonds that could form in the [d(CGCAAATTTGCG)]2 dodecamer. The dynamics of adenine amino groups in both dodecamers were also characterized using 15N T1 and 1H–15N NOE measurements. The data are consistent with the presence of internal motions for adenine amino groups. The motions are highly restricted, with an order parameter S2 of 0.8, and occur on the picosecond time‐scale. The internal motions of the three adenine amino groups in [d(CGCAAATTTGCG)]2 are similar and parallel that of the amino group in the single adenine in [d(CGCGAGCTCGCG)]2.

Structure and mechanism of LGK: insights into the bioconversion of levoglucosan

Lignocellulosic biomass is an abundant source of carbohydrates that can be used for the production of biofuels. Additional processing of biomass-derived sugars arises from the fact that the most abundant sugar product from biomass pyrolysis is the unusual anhydroring containing sugar, levoglucosan (LG). LG does not fall within the native substrate range of commonly used biocatalysts such as Escherichia coli and Saccharomyces cerevisiae. However, LG can be further converted to glucose-6-phosphate through the activity of the sugar kinase known as levoglucosan kinase (LGK), which has been isolated from a variety of fungal sources. Integration of recombinant LGK from Lipomyces starkeyi into an engineered ethanologenic Escherichia coli strain has been shown to allow for the utilization of LG as the sole carbon source for ethanol production [1]. However, challenges associated with effective utilization of LG include a high Km of LGK for LG, and the relatively low activity of LGK at physiological pH. In addition to the practical applications of LGK for biofuel production, the enzyme is an exceptional target for structural and mechanistic studies since it appears to possess dual hydrolase and kinase functionality. In order to gain a better understanding of the structure and mechanism of LGK, we have crystallized and determined several high-resolution X-ray structures of Lipomyces starkeyi LGK bound to reaction substrates and products. We have also recently collected low-resolution neutron diffraction for an LGK crystal, and further optimization of LGK crystals is currently underway to improve crystal size and diffraction. Neutron diffraction will reveal the protonation states of key residues in the active site of LGK and provide highly detailed information about hydrogen bonds, including water-bonding interactions. The rational design of new LGK constructs will be used to improve applications of this enzyme towards levoglucosan derived biofuel production.