PHYSICAL VARIABLES AS A CONTROL OF ENZYME STEREOselectivity
R. Chaloupková1, Z. Prokop1, M. Straková1, T. Mozga1 and J. Damborský1
1Loschmidt Laboratories, Faculty of Science, Masaryk University, 625 00 Brno
The applicability of enzymes in an industrial processes for the production of fine chemicals requires that a number of criteria are fulfilled. Enantioselectivity is the most important when application in asymmetric biocatalysis is being considered. Many enzymes do not provide products of high stereomeric purity under physiological conditions. The manipulation of the physical environment is attractive way to provide an additional level of control of enzyme stereochemistry. In this work, the effect of temperature and pH on enzyme structure stability, activity and enantioselectivity was investigated. Haloalkane dehalogenase DbjA from Bradyrhizobium japonicum USDA110  was selected as a model enzyme. The DbjA dehalogenase exhibits novel substrate specificity  and more importantly it is the first reported enantioselective haloalkane dehalogenase. High enantioselectivity of this enzyme was observed in reaction with selected b-substituted bromoalkanes and brominated esters .
Activity, enantioselectivity and conformational behavior of DbjA have been evaluated at different temperatures and pH. Conformational changes have been assessed by circular dichroism spectroscopy. Determined melting temperature Tm was 47.3 ± 0.2 °C. The DbjA enzyme is highly tolerant to pH changes. The secondary structure of DbjA was not significantly affected by pH in the range 5.3-10.3. At highly acidic conditions (pH<5.3), the enzyme aggregates, whereas at extreme alkaline conditions (pH>10.3) exist in predominantly disordered conformation. The temperature and pH optima of DbjA enzyme were determined with 1-iodohexane by activity measurement. Maximal activity was detected at 50 °C and at pH 9.7. The highest E value (174) of DbjA enzyme towards 2-bromopentane was determined at 20°C. The effect of pH on enantiodiscrimination of 2-bromopentane by DbjA was not observed, while the temperature significantly altered enzyme enantioselectivity. Lowering of temperature from 50 to 20°C results in thirteen times higher DbjA enantioselectivity towards 2-bromopentane. Racemic temperature as well as differential activation parameters enthalpy and entropy have been calculated based on relations between enantioselectivity and temperature. Racemic temperature was evaluated to be 85.6 °C, DR-SDHto be -69.5 kJ/mol and DR-SDSto be 0.2 kJ/mol. The enzyme preferentially catalyzed enthalpically favored (R)-enantiomer.
These observations demonstrate that haloalkane dehalogenase DbjA possess high activity and structural stability through wide range of pH conditions. Furthermore, the enantioslelectivity of DbjA enzyme with selected substrate could be improved by decreasing experimental temperature. We conclude that physical variables represent an important and easiest approach for modulation of enzyme catalytic properties which can be explored in biotechnologies.
 Y. Sato, M. Monincová, R. Chaloupková, Z. Prokop, Y. Ohtsubo, K. Minamisawa, M. Tsuda, J. Damborský & Y. Nagata, Applied and Environmental Microbiology 71 (2005) 4372-4379.
 Z. Prokop, Y. Sato, T. Mozga, R. Natsume, D.B. Janssen, M. Tsuda, Y. Nagata, T. Senda & J. Damborsky, Nature Biological Chemistry (2006) submitted.