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ABSTRACT: The special chemical and biological features of beta-peptides have been investigated intensively during recent years. Many studies emphasize the restricted biodegradability and the high metabolic stability of this class of compounds. beta-Peptidyl aminopeptidases form the first family of enzymes that hydrolyze a variety of short beta-peptides and beta-amino-acid-containing peptides. All representatives of this family were isolated from Gram-negative bacteria. The substrate specificities of the peptidases vary greatly, but the enzymes have common structural properties, and a similar reaction mechanism can be expected. This review gives an overview on the beta-peptidyl aminopeptidases with emphasis on their biochemical and structural properties. Their possible physiological function is discussed. Functionally and structurally related enzymes are compared to the beta-peptidyl aminopeptidases.
Applied Microbiology and Biotechnology 05/2007; 74(6):1197-204. · 3.42 Impact Factor
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ABSTRACT: In most cases, enantiomers of chiral compounds behave differently in biochemical processes. Therefore, the effects and the environmental fate of the enantiomers of chiral pollutants need to be investigated separately. In this review, the different fates of the enantiomers of chiral phenoxyalkanoic acid herbicides, acetamides, organochlorines, and linear alkylbenzenesulfonates are discussed. The focus lies on biological degradation, which may be enantioselective, in contrast to non-biotic conversions. The data show that it is difficult to predict which enantiomer may be enriched and that accumulation of an enantiomer is dependent on the environmental system, the species, and the organ. Racemization and enantiomerization processes occur and make interpretation of the data even more complex. Enantioselective degradation implies that the enzymes involved in the conversion of such compounds are able to differentiate between the enantiomers. "Enzyme pairs" have evolved which exhibit almost identical overall folding. Only subtle differences in their active site determine their enantioselectivities. At the other extreme, there are examples of non-homologous "enzyme pairs" that have developed through convergent evolution to enantioselectively turn over the enantiomers of a chiral compound. For a better understanding of enantioselective reactions, more detailed studies of enzymes involved in enantioselective degradation need to be performed.
Applied Microbiology and Biotechnology 05/2004; 64(3):300-16. · 3.42 Impact Factor
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ABSTRACT: In most cases, enantiomers of chiral compounds behave differently in biochemical processes. Therefore, the effects and the environmental fate of the enantiomers of chiral pollutants need to be investigated separately. In this review, the different fates of the enantiomers of chiral phenoxyalkanoic acid herbicides, acetamides, organochlorines, and linear alkylbenzenesulfonates are discussed. The focus lies on biological degradation, which may be enantioselective, in contrast to non-biotic conversions. The data show that it is difficult to predict which enantiomer may be enriched and that accumulation of an enantiomer is dependent on the environmental system, the species, and the organ. Racemization and enantiomerization processes occur and make interpretation of the data even more complex. Enantioselective degradation implies that the enzymes involved in the conversion of such compounds are able to differentiate between the enantiomers. Enzyme pairs have evolved which exhibit almost identical overall folding. Only subtle differences in their active site determine their enantioselectivities. At the other extreme, there are examples of non-homologous enzyme pairs that have developed through convergent evolution to enantioselectively turn over the enantiomers of a chiral compound. For a better understanding of enantioselective reactions, more detailed studies of enzymes involved in enantioselective degradation need to be performed.
Applied Microbiology and Biotechnology 03/2004; 64(3):300-316. · 3.42 Impact Factor
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Journal of Biological Chemistry 274 (1999).
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In: Flavins and flavoproteins 1999 : Proceedings 13th International Symposium, Konstanz, Germany, 1999 / Ghisla S, Kroneck P, Macheroux P, Sund H, (Eds). - Berlin : Agency for Scientific Publications, 1999.
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ABSTRACT: The substrate reactivity of the flavoenzyme 2-hydroxybiphenyl 3-monooxygenase (EC 1.14.13.44, HbpA) was changed by directed evolution using error-prone PCR. In situ screening of mutant libraries resulted in the identification of proteins with increased activity towards 2-tert-butylphenol and guaiacol (2-methoxyphenol). One enzyme variant contained amino acid substitutions V368A/L417F, which were inserted by two rounds of mutagenesis. The double replacement improved the efficiency of substrate hydroxylation by reducing the uncoupled oxidation of NADH. With guaiacol as substrate, the two substitutions increased Vmax from 0.22 to 0.43 units mg1 protein and decreased the K'm from 588 to 143 ?M, improving k'cat/K'm by a factor of 8.2. With 2-tert-butylphenol as the substrate, k'cat was increased more than 5-fold. Another selected enzyme variant contained amino acid substitution I244V and had a 30␑igher specific activity with 2-sec-butylphenol, guaiacol, and the "natural" substrate 2-hydroxybiphenyl. The K'm for guaiacol decreased with this mutant, but the K'm for 2-hydroxybiphenyl increased. The primary structure of HbpA shares 20.1␜equence identity with phenol 2-monooxygenase from Trichosporon cutaneum. Structure homology modeling with this three-domain enzyme suggests that Ile244 of HbpA is located in the substrate binding pocket and is involved in accommodating the phenyl substituent of the phenol. In contrast, Val368 and Leu417 are not close to the active site and would not have been obvious candidates for modification by rational design.
Journal of Biological Chemistry 277 (2002). - ISSN 0021-9258.
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ABSTRACT: Incubation of resting cells of Sphingobium indicum B90A, Sphingobium japonicum UT26, and Sphingobium francense Sp+ showed that they were able to transform ß- and -hexachlorocyclohexane (ß- and -HCH, respectively), the most recalcitrant hexachlorocyclohexane isomers, to pentachlorocyclohexanols, but only resting cells of strain B90A could further transform the pentachlorocyclohexanol intermediates to the corresponding tetrachlorocyclohexanediols. Moreover, experiments with resting cells of Escherichia coli expressing the LinB proteins of strains B90A, UT26, and Sp+ indicated that LinB was responsible for these transformations. Purified LinB proteins from all three strains also effected the formation of the respective pentachlorocyclohexanols. Although the three LinB enzymes differ only marginally with respect to amino acid sequence, they showed interesting differences with respect to substrate specificity. When LinB from strain B90A was incubated with ß- and -HCH, the pentachlorocyclohexanol products were further transformed and eventually disappeared from the incubation mixtures. In contrast, the LinB proteins from strains UT26 and Sp+ could not catalyze transformation of the pentachlorocyclohexanols, and these products accumulated in the incubation mixture. A mutant of strain Sp+ lacking linA and linB did not degrade any of the HCH isomers, including ß-HCH, and complementation of this mutant by linB from strain B90A restored the ability to degrade ß- and -HCH.
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M. Suar,
A. Hauser,
T Poiger,
H R Buser,
M D Müller,
C. Dogra,
V. Raina,
C. Holliger,
J R van der Meer,
R Lal, H.-P. E. Kohler
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ABSTRACT: Sphingomonas paucimobilis B90A contains two variants, LinA1 and LinA2, of a dehydrochlorinase that catalyzes the first and second steps in the metabolism of hexachlorocyclohexanes (R. Kumari, S. Subudhi, M. Suar, G. Dhingra, V. Raina, C. Dogra, S. Lal, J. R. van der Meer, C. Holliger, and R. Lal, Appl. Environ. Microbiol. 68:6021-6028, 2002). On the amino acid level, LinA1 and LinA2 were 88% identical to each other, and LinA2 was 100% identical to LinA of S. paucimobilis UT26. Incubation of chiral -hexachlorocyclohexane (-HCH) with Escherichia coli BL21 expressing functional LinA1 and LinA2 S-glutathione transferase fusion proteins showed that LinA1 preferentially converted the (+) enantiomer, whereas LinA2 preferred the (–) enantiomer. Concurrent formation and subsequent dissipation of ß-pentachlorocyclohexene enantiomers was also observed in these experiments, indicating that there was enantioselective formation and/or dissipation of these enantiomers. LinA1 preferentially formed (3S,4S,5R,6R)-1,3,4,5,6-pentachlorocyclohexene, and LinA2 preferentially formed (3R,4R,5S,6S)-1,3,4,5,6-pentachlorocyclohexene. Because enantioselectivity was not observed in incubations with whole cells of S. paucimobilis B90A, we concluded that LinA1 and LinA2 are equally active in this organism. The enantioselective transformation of chiral -HCH by LinA1 and LinA2 provides the first evidence of the molecular basis for the changed enantiomer composition of -HCH in many natural environments. Enantioselective degradation may be one of the key processes determining enantiomer composition, especially when strains that contain only one of the linA genes, such as S. paucimobilis UT26, prevail.
