Quantitative Analysis of Substrate Specificity of Haloalkane Dehalogenase LinB from Sphingomonas paucimobilis UT26

Abstract

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 K(m) and k(cat) were determined for 25 substrates that showed detectable cleavage by the enzyme and low abiotic hydrolysis. Classical quantitative structure-activity relationships (QSARs) were used to correlate the kinetic constants with molecular descriptors and resulted in a model that explained 94% of the 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 nonlinearly 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 the 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 as follows: (i) first-shell residues Asn38, Asp108, Trp109, Glu132, Ile134, Phe143, Phe151, Phe169, Val173, Trp207, Pro208, Ile211, Leu248, and His272, (ii) tunnel residues Pro144, Asp147, Leu177, and Ala247, and (iii) 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 toward a different specificity is discussed on the basis of quantitative comparison of models derived for two protein family members.

Haloalkane Dehalogenase LinB Is Responsible for β- and δ-Hexachlorocyclohexane Transformation inSphingobium indicumB90A

“…A close analysis revealed that the LinB enzymes of Spϩ and UT26 differ from each other by three amino acids and that LinB of B90A differs from those of Spϩ and UT26 by six and seven amino acids residues, respectively. These differences in amino acids are outside the putative catalytic domain (D-108, H-272, and E-132) (7,11,17,18,22,27,28,33,34,37,40,47), but they seem to play an important role in the substrate specificity of the enzymes. Although the studies with purified LinB proteins from B90A and Spϩ are preliminary, they strongly indicate that strain B90A is best suited for biodegradation of ␤-HCH, the most recalcitrant isomer.…”

Biochemistry of Microbial Degradation of Hexachlorocyclohexane and Prospects for Bioremediation

“…The major catalytic domain of LinB belongs to the large and well-characterized ␣/␤-hydrolase fold superfamily of proteins, which characteristically carry out two-step hydrolytic reactions driven by a nucleophile (Asp) that is part of a catalytic triad and using an oxanion hole to stabilize the intermediate (69,114,117). LinB has been the subject of several crystallographic (92,123,166) and computational studies (32,33,69,122,130).…”

Weak Activity of Haloalkane Dehalogenase LinB with 1,2,3-Trichloropropane Revealed by X-Ray Crystallography and Microcalorimetry

“…LinB is an enzyme involved in a biochemical pathway for the degradation of the pesticide lindane (␥-hexachlorocyclohexane), where it catalyzes the conversion of 1,3,4,6-tetrachloro-1,4-cyclohexadiene to 2,4,5-trichloro-2,5-cyclohexadiene-1-ol (16). A wide spectrum of haloalkanes can be converted by LinB; however, TCP was estimated to be a haloalkane that did not serve as a substrate for LinB when conventional analytical methods were used (6,12). We decided to apply X-ray crystallography to determine the LinB-TCP complex structure and reveal the structural basis for inactivity.…”