PubMed · 13935399
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J B MIALE, A R WINNINGHAM, J W KENT. 1963-01-26. Staphylococcal isocoagulases.. https://doi.org/10.1038/197392a0
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One-nanosecond molecular dynamics trajectories of three haloalkane dehalogenases (DhlA, LinB, and DhaA) are compared. The main domain was rigid in all three dehalogenases, whereas the substrate specificity-modulating cap domains showed considerably higher mobility. The functionally relevant motions were spread over the entire cap domain in DhlA, whereas they were more localized in LinB and DhaA. The highest amplitude of essential motions of DhlA was noted in the alpha4'-helix-loop-alpha4-helix region, formerly proposed to participate in the large conformation change needed for product release. The highest amplitude of essential motions of LinB and DhaA was observed in the random coil before helix 4, linking two domains of these proteins. This flexibility is the consequence of the modular composition of haloalkane dehalogenases. Two members of the catalytic triad, that is, the nucleophile and the base, showed a very high level of rigidity in all three dehalogenases. This rigidity is essential for their function. One of the halide-stabilizing residues, important for the catalysis, shows significantly higher flexibility in DhlA compared with LinB and DhaA. Enhanced flexibility may be required for destabilization of the electrostatic interactions during the release of the halide ion from the deeply buried active site of DhlA. The exchange of water molecules between the enzyme active site and bulk solvent was very different among the three dehalogenases. The differences could be related to the flexibility of the cap domains and to the number of entrance tunnels.
The diastereomers of beta-methyl-L-kynurenine were prepared by preparative ozonolysis of the respective diastereomers of beta-methyl-L-tryptophan. A practical method for preparative enzymatic resolution of the diastereomers of beta-methyltryptophan was developed using carboxypeptidase A digestion of the N-trifluoroacetyl derivatives. The stereochemical assignment was confirmed by X-ray crystal structure determination of (2S, 3R)-threo-beta-methyl-L-tryptophan. (2S,3S)-erythro-beta-Methyl-L-kynurenine is a slow substrate for kynureninase from Pseudomonas fluorescens (k(cat)/K(m) = 0.1% that of L-kynurenine), producing anthranilic acid, while (2S,3R)-threo-L-kynurenine is about 390-fold less reactive than erythro. Rapid-scanning stopped-flow measurements show that beta-methyl substitution affects the rate of alpha-deprotonation of the L-kynurenine-pyridoxal-5'-phosphate Schiffs base. This is consistent with the stereoelectronic requirements of the reaction. These results are the first demonstration that beta-substituted kynurenines can be substrates for kynureninase, and may be useful in the design of mechanism-based inhibitors.
Trihalogenated propanes are toxic and recalcitrant organic compounds. Attempts to obtain pure bacterial cultures able to use these compounds as sole carbon and energy sources were unsuccessful. Both the haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 (DhlA) and that from Rhodococcus sp. strain m15-3 (DhaA) were found to dehalogenate trihalopropanes to 2,3-dihalogenated propanols, but the kinetic properties of the latter enzyme are much better. Broad-host-range dehalogenase expression plasmids, based on RSF1010 derivatives, were constructed with the haloalkane dehalogenase from Rhodococcus sp. strain m15-3 under the control of the heterologous promoters P(lac), P(dhlA), and P(trc). The resulting plasmids yielded functional expression in several gram-negative bacteria. A catabolic pathway for trihalopropanes was designed by introducing these broad-host-range dehalogenase expression plasmids into Agrobacterium radiobacter AD1, which has the ability to utilize dihalogenated propanols for growth. The recombinant strain AD1(pTB3), expressing the haloalkane dehalogenase gene under the control of the dhlA promoter, was able to utilize both 1,2,3-tribromopropane and 1,2-dibromo-3-chloropropane as sole carbon sources. Moreover, increased expression of the haloalkane dehalogenase resulted in elevated resistance to trihalopropanes.