Role of solvents in the control of enzyme selectivity in organic media

Carrea, Giacomo; Ottolina, Gianluca; Riva, Sergio

doi:10.1016/s0167-7799(00)88907-6

Cited by 310 publications

(117 citation statements)

References 42 publications

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“…Because subtilisin exhibits markedly different catalytic activity and other properties in different solvents (7)(8)(9)(10)(11)(12)(13)(14)(21)(22)(23)(24), the structure of the active site in these solvents is a critical issue. We find that not only is the overall structure of the crystalline acyl-enzyme the same whether formed in acetonitrile or in water, but it can be seen in Fig.…”

Section: Resultsmentioning

confidence: 99%

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Comparison of x-ray crystal structures of an acyl-enzyme intermediate of subtilisin Carlsberg formed in anhydrous acetonitrile and in water

Schmitke

Stern

Klibanov

1998

Proc. Natl. Acad. Sci. U.S.A.

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The x-ray crystal structures of trans-cinnamoyl-subtilisin, an acyl-enzyme covalent intermediate of the serine protease subtilisin Carlsberg, have been determined to 2.2-Å resolution in anhydrous acetonitrile and in water. The cinnamoyl-subtilisin structures are virtually identical in the two solvents. In addition, their enzyme portions are nearly indistinguishable from previously determined structures of the free enzyme in acetonitrile and in water; thus, acylation in either aqueous or nonaqueous solvent causes no appreciable conformational changes. However, the locations of bound solvent molecules in the active site of the acyl-and free enzyme forms in acetonitrile and in water are distinct. Such differences in the active site solvation may contribute to the observed variations in enzymatic activities. On prolonged exposure to organic solvent or removal of interstitial solvent from the crystal lattice, the channels within enzyme crystals are shown to collapse, leading to a drop in the number of active sites accessible to the substrate. The mechanistic and preparative implications of our findings for enzymatic catalysis in organic solvents are discussed.Enzymes in organic solvents, instead of their natural aqueous milieu, remain synthetically useful catalysts (1-6). In addition, they display remarkable properties, e.g., solvent-dependent selectivity (7,8). Such solvent dependences of prochiral selectivity (9) and enantioselectivity (10) of crystalline enzymes in nonaqueous media have been nearly quantitatively predicted using structure-based molecular modeling and thermodynamic calculations.Despite recent advances (11)(12)(13)(14), the question of why enzymatic activity is often much reduced in organic solvents compared with water is far from resolved. Such an understanding, like that concerning the selectivity, requires both structural and mechanistic information. Recently, structures of several enzymes in neat organic solvents, namely those of subtilisin Carlsberg in acetonitrile (15, 16) and dioxane (17), ␥-chymotrypsin in hexane (18,19), and elastase in acetonitrile (20), have been elucidated and found to be essentially the same as in water. Furthermore, the structures in hexane of a ␥-chymotrypsin-product complex has been determined (19) and that of a tetrahedral complex claimed (18). However, to provide further insight into the enzyme mechanism in organic solvents, structures of covalent reaction intermediates should be determined. To the best of our knowledge, no such structures have been available, until now.All the aforementioned enzyme structures are of serine proteases, for which the universal reaction intermediate in water is an acyl-enzyme. In organic solvents, however, the formation of such an intermediate in the catalysis by subtilisin is supported by kinetic but not direct structural data (21-24).Moreover, it is simply presumed that the structure of the enzyme-substrate intermediate formed in different solvents is the same (9, 10). The most direct way to experimentally test this assumpti...

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Section: Resultsmentioning

confidence: 99%

“…In addition, they display remarkable properties, e.g., solvent-dependent selectivity (7,8). Such solvent dependences of prochiral selectivity (9) and enantioselectivity (10) of crystalline enzymes in nonaqueous media have been nearly quantitatively predicted using structure-based molecular modeling and thermodynamic calculations.…”

Section: Introductionmentioning

confidence: 99%

Comparison of x-ray crystal structures of an acyl-enzyme intermediate of subtilisin Carlsberg formed in anhydrous acetonitrile and in water

Schmitke

Stern

Klibanov

1998

Proc. Natl. Acad. Sci. U.S.A.

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“…Besides influencing stability,the reaction medium can also modify enzyme selectivity. [15][16] Thee ffect of cosolvents (Table S5) was determined by using 2-butanone,t he conversion of which to two possible regioisomers is of industrial interest (Scheme S1). [17] All reactions were stopped after 48 h at 17 8 8C.…”

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confidence: 99%

Characterization and Crystal Structure of a Robust Cyclohexanone Monooxygenase

et al. 2016

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Cyclohexanone monooxygenase (CHMO) is ap romising biocatalyst for industrial reactions owing to its broad substrate spectrum and excellent regio-, chemo-, and enantioselectivity.However,the lowstability of many BaeyerVilliger monooxygenases is an obstacle for their exploitation in industry.C haracterization and crystal structure determination of ar obust CHMO from Thermocrispum municipale is reported. The enzyme efficiently converts avariety of aliphatic, aromatic,a nd cyclic ketones,a sw ell as prochiral sulfides.A compact substrate-binding cavity explains its preference for small rather than bulky substrates.S mall-scale conversions with either purified enzyme or whole cells demonstrated the remarkable properties of this newly discovered CHMO.T he exceptional solvent tolerance and thermostability make the enzyme very attractive for biotechnology.

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“…Medium engineering refers to the rational manipulation of the reaction medium to positively influence the properties of the enzyme with respect to the reaction of synthesis (Clapés et al 1990a;Clapés et al 1990b;Khmelnitsky et al 1991;Ryu and Dordick, 1992;Wescott and Klibanov, 1994;Carrea et al 1995, Chaudhary et al 1996. This frequently implies the substitution of the usual aqueous medium for a non conventional medium in which water has been replaced partially or almost totally by another solvent (Hari Krishna, 2002).…”

Section: Medium Engineeringmentioning

confidence: 99%

Peptide synthesis: chemical or enzymatic

Guzmán

Barberis

Illanes

2007

Electron. J. Biotechnol.

169

139

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Abbreviations:CD 280synthesis is probably the way to go, since the good properties of each technology can be synergistically used in the context of one process objective.Peptides are heteropolymers composed by amino acid residues linked by peptidic bonds between the carboxyl group of one amino acid residue and the α-amino group of the next one. The definition is rather vague in terms of chain length, peptides ranging from two to a few dozens residues. Its lower limit of molecular mass has been set rather arbitrarily in 6000 Da; molecules larger than that are considered proteins. Peptides are molecules of paramount importance in several fields, especially in health care and nutrition. The case of the hormone insulin (51 residues, 5773 Da) and the non-caloric sweetener aspartame (a dipeptide of aspartic acid and esterified phenylalanine) are relevant examples of those fields of application and the range of molecular size. Medium to small size peptides are, however, the most relevant for such applications.

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Role of solvents in the control of enzyme selectivity in organic media

Cited by 310 publications

References 42 publications

Comparison of x-ray crystal structures of an acyl-enzyme intermediate of subtilisin Carlsberg formed in anhydrous acetonitrile and in water

Comparison of x-ray crystal structures of an acyl-enzyme intermediate of subtilisin Carlsberg formed in anhydrous acetonitrile and in water

Characterization and Crystal Structure of a Robust Cyclohexanone Monooxygenase

Peptide synthesis: chemical or enzymatic

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