Quantitative Analysis of Substrate Specificity of Haloalkane Dehalogenase LinB from Sphingomonas paucimobilis UT26
Kmunicek, J., Hynkova, K., Jedlicka, T., Nagata, Y., Negri, A., Gago, F., Wade, R.C., Damborsky, J.
BIOCHEMISTRY 44: 3390-3401 (2005)
Haloalkane dehalogenases are microbial enzymes that cleave a carbon-halogen bond in halogenated compounds. The haloalkane dehalogenase LinB, isolated from Sphingomonas paucimobilis UT26, is a broad-specificity enzyme. Fifty five halogenated aliphatic and cyclic hydrocarbons were tested for dehalogenation with the LinB enzyme. The compounds for testing were systematically selected using a statistical experimental design. Steady-state kinetic constants Km and kcat were determined for twenty five substrates that showed detectable cleavage by the enzyme and low abiotic hydrolysis. Classical Quantitative Structure-Activity Relationships (QSAR) were used to correlate the kinetic constants with molecular descriptors and resulted in a model that explained 94% of experimental data variability. The binding affinity of the tested substrates for this haloalkane dehalogenase correlated with hydrophobicity, molecular surface, dipole moment and volume/surface ratio. Binding of the substrate molecules in the active site pocket of LinB depends non-linearly on the size of the molecules. Binding affinity increases with increasing substrate size up to a chain length of six carbon atoms and then decreases. Comparative binding energy (COMBINE) analysis was then used to identify amino acid residues in LinB that modulate its substrate specificity. A model with three statistically significant principal components explained 95% of experimental data variability. Van der Waals interactions between substrate molecules and the enzyme dominated the COMBINE model, in agreement with the importance of substrate size in the classical QSAR model. Only a limited number of protein residues (6-8%) contribute significantly to the explanation of variability in binding affinities. The amino acid residues important for explaining variability in binding affinities are: (i) the first shell residues—Asn38, Asp108, Trp109, Glu132, Ile134, Phe143, Phe151, Phe169, Val173, Trp207, Pro208, Ile211, Leu248, and His272, (ii) the tunnel residues—Pro144, Asp147, Leu177, and Ala247, and (iii) the second shell residues—Pro39 and Phe273. The tunnel and the second shell residues represent the best targets for modulating specificity since their replacement does not lead to loss of functionality by disruption of the active site architecture. The mechanism of molecular adaptation towards different specificity is discussed based on quantitative comparison of models derived for two protein family members.