CATALYTIC PEPTIDES CONSISTING OF A DESIGNED HELIX-LOOP-HELIX MOTIF AND THEIR USE

Abstract: A catalytic peptide consisting of a designed helix-loop-helix motif, consisting of at least 37 amino acids, and most preferably of 42 amino acids, with lysine, arginine, histidine, glutamic acid, or aspartic acid in positions 8, 10, 11, 15, 19, 23, 26, 30, 34and 37. Said peptide should preferably also have leucine in positions 12 and 31, isoleucine in position 9, phenylalanine in positions 35 and 38, and norleucine or leucine in positions 5, 16 and 27. Catalytic molecules and catalytic proteins comprising the catalytically active amino acids of the catalytic peptide according to the invention or the whole catalytic peptide. Use of such catalytic substances in combination with pyridoxal phosphate. Catalyst for amino acid synthesis comprising two dimerised catalytic peptides according to the invention and pyridoxal phosphate.

Description: CATALYTIC PEPΗDES CONSISTING OF A DESIGNED HELIX-LOOP-HELIX MOΗF AND THEIR USE Technical field of the invention The present invention relates to new catalytic pep- tides and catalysts for ammo acid synthesis. Background art De novo designed folded polypeptides provide a large pool of new shapes, new functions and new materials. An important area is the design of new catalysts that catalyse reactions not performed by nature. There exists some catalysts for amino acid synthesis, however most of them have some disadvantages, e.g. some of them do not give high enough reaction rates, while others are not possible to use for the production of enantiomeπcally pure amino acids. In particular, the naturally occurring enzymes cannot be used for the synthesis of non-natural ammo acids. The inventor of the present invention has, together with co-workers, earlier in two articles, J. Chem. Soc. Perkin. Trans. 2, 1998, 10, 2271-2274 and Chem. Commun., 1998, 15, 1547-1548, described folded polypeptide motifs, one for decarboxylation of oxaloacetate and one for use in mimicking enzymatic transammation. However, these folded polypeptides differs in many ways from the catalytic peptides according to the invention. Most lmpor- tantly the peptide described the latter publication has only demonstrated an ability to bind the cofactor pyridoxal phosphate m a non-productive mode, i.e. one where the cofactor forms an lmine with a lysine side chain. In order to catalyse the transammation reaction the cofactor must release the lysine side chain and form such a bond with the ammo group of the ammo acid to be deammated. For that reason, the peptide reported in Chem. Comm. has not demonstrated the key function of the transammation catalysts of the present invention, i.e. the ability to to bind the the intermediate, formed from the ammo acid and the cofactor, by non-covalent forces m a conformation where residues m the sequence of the catalyst, exposed on the surface of the folded peptide, catalyses the chemical transformations. Summary of the invention The object of the present invention is to provide a new catalytic system specially designed for the catalysis of ammo acid synthesis, particularly synthesis of amino acids not found in nature, said system being more efficient than the ones existing today. More precisely, the invention provides new catalytic peptides and catalysts suitable for catalysis of the transammation and/or de- carboxylation reactions in synthesis of ammo acids. Furthermore, the catalysts according to the invention can be designed to have high specificity. The present invention thus relates to a new catalytic peptide consisting of a designed helix-loop-helix motif, and to catalysts comprising two dimerised peptides of this kind. One advantage of the present invention is the ability of the catalytic peptides according to the invention to bind key intermediates along the reaction pathway by non-covalent bonds and thus catalysing the transamnation reaction. Another advatage of the peptides of the present invention in comparison with prior art peptides, and especially those reported in the publications mentioned as in the background part, is that the peptide catalysts reported here show enhanced and synergistic activity when used m the forms of heterodimers, i.e. when both the de- carboxylase mimetic and the transammase mimetic are used simultaneously (further illustrated m figure 6 B) . Ki- netic evidence shows that synergistic effects are obtained between the two, and that heterodimers are more efficient catalysts. The characterising features of the invention will be evident from the following description and the appended claims . Detailed description of the invention The catalytic peptide according to the invention thus consists of a helix-loop-helix motif, consisting of at least 37 ammo acids and have lysine (Lys) , arginine (Arg) , histidine (His) , glutamic acid (Glu) , or aspartic acid (Asp), independently of each other, m positions 8, 10, 11, 15, 19, 23, 26, 30, 34, and 37. Lys and Arg are particularly suitable for production of acid ammo acids, while Glu and Asp m combination with His, Arg or Lys are particularly suitable for production of basic ammo ac- ids. These ammo acids are exposed on the surface of the motif. Ammo acids with uncharged side chains are also produced by these peptides. A preferred catalytic peptide according to the invention consists of 42 ammo acids. Preferably the ammo acids in positions 12 and 31 are leucine (Leu) , in position 9 isoleucine (lie) , in positions 35 and 38 phenylalanine (Phe) , and in positions 5, 16 and 27 norleucine (Nle) . However, Nle can be replaced by Leu without loss of activity. During the research work leading to the pre- sent invention, it was found that this combination and the relative positions of these ammo acids results in an arrangement of the above described surface ammo acids in a defined spatial structure leading to a three dimensional motif with the catalytic activity according to the invention. A preferred embodiment of the catalytic peptide according to the invention thus has a sequence according to SEQ ID NO: 1 given m the appended sequence listing. In an even more preferred embodiment, the N- terminal and/or the C-termmal of the peptide are "capped", which herein means that the ammo group of the ammo acid at the N-termmal is acetylated, which is denoted m the se- quences by Ac, and that the carboxy group of the am o acid at the C-termmal is amidated, which is denoted m the sequences by NH 2 . The general sequence of a capped peptide according to the invention is SEQ ID NO: 2 given the appended sequence listing. Two especially preferred catalytic peptides according to the invention are the peptides with SEQ ID NO : 3 and SEQ ID NO : 5 given in the appended sequence listing, and the "capped" version of these peptides, i.e. the pep- tides with SEQ ID NO: 4 and SEQ ID NO: 6 given in the appended sequence listing. The peptides according to the invention may e.g. be produced with conventional chemical synthesis or by genetic engineering, e.g. m a host such as E. coll. The invention also relates to catalytic molecules and proteins comprising the above mentioned sequences. Such a catalytic substance may be the result of incorporation of the catalytically active ammo acids, i.e. the histidmes, lysmes, argm es, glutamic acids, and/or aspartic acids in positions 8, 10, 11, 15, 19, 23, 26, 30, 34, and 37, into a larger molecule, preferably a naturally occurring protein, m such a way that the ammo acid in those positions retain the same spatial geometry as they have m the catalytic peptides according to the invention. In order to obtain the desired catalytic effect of the catalytic peptides, catalytic molecules and catalytic proteins according to the invention, they should be used in combination with pyridoxal phosphate. The catalyst according to the invention comprises two catalytic peptides in the form of helix-loop-helix motifs according to the invention which are dimerised to form a four-helix bundle. In its simplest form, the catalyst according to the invention consists only of such a homodimenc four-helix bundle. As stated above, it is however also possible to incorporate the catalytic residues of the helix-loop-helix motifs according to the in- vention in a larger peptide, such as a naturally occurring protein. When this is done, it is important to retain the relative positions of the amino acid side chains of the residues in positions 8, 10, 11, 15, 19, 23, 26, 30, 34 and 37. This can be accomplished in several ways by the tools of molecular biology or peptide synthesis. For example, the whole sequence of the catalytic peptide according to the invention can be fused to a naturally occurring protein of high stability to form a so called "fusion protein", the whole sequence of the catalytic peptide according to the invention can be incorporated into a naturally occurring protein by replacing a helix- loop-helix motif that already exists. It is also possible to incorporate the catalytic residues, i.e. the amino ac- ids in positions 8, 10, 11, 15, 19, 23, 26, 30, 34, and 37 in the catalytic peptide according to the invention, in the same relative geometries in a helix-loop-helix motif in a naturally occurring protein. It is also possible to incorporate these catalytic residues into a naturally occurring protein that does not have a helix-loop-helix motif in such a way that the relative geometries of the residues in the catalytic helix-loop-helix motif are retained. The catalyst according to the invention may also be a non-peptidic molecule wherein the functional groups of the side chains of the catalytic residues in said positions are in the same relative positions as in the catalytic peptide according to the invention. Such a catalyst will include amino groups, guanidino groups and imidazoyl groups in the same relative positions as the side chains of lysines, arginines and histidines in the helix-loop-helix motif according to SEQ. ID No. 1. The catalyst according to the invention may function as a transaminase mimetic, i.e. as a catalyst of a transammation reaction. A preferred embodiment of a transami- nation catalyst according to the invention is a dimerised four-helix bundle consisting of two helix-loop-helix motifs, the sequence of each being SEQ. ID NO. 3 or . The efficiency of the catalyst can be further enhanced by replacing one of the transaminase mimetic motifs with a second catalytic peptide functioning as a decarboxylase mimetic, i.e. as a catalyst of a decarboxylation reac- tion. It is thus possible two use two different catalytic peptides according to the invention in order to consecutively perform the transaminase reaction and decarboxylation reaction. A preferred embodiment of a catalyst according to the invention functioning both as a transami- nase mimetic and a decarboxylase mimetic is a dimerised four-helix bundle consisting of one motif, the sequence of which is SEQ. ID NO. 3 or 4 , and one motif, the sequence of which is SEQ. ID NO. 5 or 6 , which is further illustrated below. In a further embodiment of the inven- tion the decarboxylase function may be incorporated into the helix-loop-helix motif that catalyses the transammation reaction. This catalyst will provide efficient transamination to continuously regenerate the pyridoxamine phosphate needed for the catalytic amination of α-keto acids to form amino acids . The production of many amino acids is based on the use of the pyridoxal phosphate cofactor in the transamination reaction. When the catalysts according to the in- vention are used, it is possible to use the naturally occurring form of the pyridoxal phosphate cofactor instead of modified, synthetic versions of this cofactor. This feature is an important advantage or the invention because the synthesis of such derivatives for commercial use is often expensive and such cofactors may not be able to find the optimum geometry for transamination with optimum efficiency. The binding of the pyridoxal phosphate cofactor and intermediates in pyridoxal phosphate mediated reaction have been shown in the present invention to be controlled by the non-covalent interactions between the phosphate group of the cofactor, other charged substituents of the pyridoxal phosphate derived intermedi- ates and arginine or lysine residues on the surface of the folded motif. A key feature of the present invention is thus the ability of the catalytic peptides to bind the aldimine and ketimme intermediates and to catalyse stereospecifically the 1,3 -proton transfer that mtercon- verts these intermediates. In order to accomplish the binding of these intermediates the transaminase mimetic according to the invention is rich in argmmes and lysmes that bind the m- termediates purely by non-covalent forces. In order to accomplish the 1,3 -proton transfer reaction the transamination catalyst has histidine and lysine residues. The invention will now be further explained m the following examples. These examples are only intended to illustrate the invention and should in no way be considered to limit the scope of the invention. Brief description of the drawings In the examples, reference is made to the accompany- ing drawings on which: Fig. la is a UV-spectrum illustrating a transammation reaction catalysed by a catalyst according to the invention. Fig. lb is a X H-NMR spectrum illustrating a transamma- tion and decarboxylation reaction catalysed by a catalyst according to the invention. Fig. 2 illustrates the coupling of the transammation and the decarboxylation reactions performed with the catalyst according to the invention. Fig. 3 illustrates the reaction mechanism for transamination of aspartic acid to oxaloacetate . Fig. 4 illustrates the reaction mechanism for decarboxylation of oxaloacetate. Fig. 5 illustrates the reaction rates for transamma- tion of aspartic acid to oxaloacetate, catalysed with a peptide according to the invention. Fig. 6 A illustrates the reactivity of the peptide with SEQ ID NO: 4 in transammation. Fig. 6 B illustrates the reactivity of the peptide with SEQ ID NO: 4 n combination with the peptide with SEQ ID NO: 6 m transammation. Fig. 7 illustrates the enantioselectivity of the peptides according to the invention in transammation. Fig. 8 is a modelled structure of the peptide with SEQ ID NO: 4. Fig. 9 is a modelled structure of the dimer formed by the peptide with SEQ ID NO: 3 and the peptide with SEQ ID NO: 6. Examples Example 1 - preparation of catalysts according to the invention A catalyst according to the invention, the peptide with SEQ ID NO: 3, was synthesised on a Fmoc-Gly-PEG-Ps polymer (PerSeptive Biosystems) using a PerSeptive Bio- systems Pioneer automated peptide synthesiser and a standard Fmoc chemistry protocol. The peptide was cleaved from the polymer and deprotected with TFA (10 ml) , an- isole (220 μl) , ethanedithiol (333 μl) and thioanisole (555 μl) for two hours at room temperature. After diethyl ether precipitation and lyophilisation the peptide was purified by reversed-phase HPLC on a semi -preparative C-8 Kromasil, 7 μm column. It was eluated isocratically using a solvent with 38% propan-2-ol in 0.1% TFA, a flow rate of 10 ml/mm and UV detection at 220 nm. The peptide was identified by electrospray mass spectrometry (VG Analytical, ZabSpec) . The obtained molecular weight was withm 1 au from the calculated and no high molecular weight lmpu- rities could be detected. The purity was estimated to be more than 95% from ES-MS. The peptide with SEQ ID NO: 5, was synthesised in the same way. The peptides with SEQ ID NO: 4 and SEQ ID NO: 6, below called peptides 4 and 6, respectively, were synthe- sised in the same way except that the final step before cleavage from the resin is an acetylation of the free N- terminal amino group by acetic anhydride. The choice of polymer linker decides whether the C-terminal is capped or not. PAL-PEG-PS was used to obtain C-terminal amides, and PAC-PEG-PS was used to obtain the free acids. A modelled structure of peptide 4 is shown in figure 8. The model shows docked aldimine intermediate and Arg, Lys and His residues involved in catalysis. Only one monomer is shown for reasons of clarity, but the folded peptide is a dimer. Example 2 A catalyst consisting of two dimerised peptides 4 was used to catalyse the transamination reaction of as- partic acid to form pyridoxamine phosphate and oxaloacetate. The transamination reaction is a multistep reversible reaction and at equilibrium the mixture of species coexist. The reaction can however be driven to completion by an oxaloacetate decarboxylase that consumes one of the reaction products, as illustrated below in example 3. The reaction between peptide 4, pyridoxal phosphate and aspartic acid was monitored by UV-spectroscopy, and the result is shown in figure la. The UV-spectrum shows the pyridoxal phosphate absorbance at 390 nm and the pyridoxamine phosphate/ketimine absorbance at 335 nm. The reaction mixture contains 0.6 mM peptide 4, 5 equivalents of pyridoxal phosphate and a 26-fold excess of aspartic acid in a Bis-Tris buffer solution at pH = 7.0 and 298 K. Example 3 In this example, the transamination reaction from example 2 was directly coupled to the decarboxylation re- action. The complete reaction scheme is illustrated in figure 2, and in more detail in figure 3, showing the reaction mechanism for transamination of aspartic acid to form oxaloacetate, together with figure 4, showing the reaction mechanism for the decarboxylation of oxalo- acetete . The oxaloacetate, i.e. the α-ketoacid of aspartic acid, produced by the transamination reaction described in example 2 is decarboxylated to form pyruvate. This re- action can be catalysed by the catalysts according to the invention. In this example, the catalyst consists of peptide 4, as the transaminase mimetic, and peptide 6, as the decarboxylase mimetic. The reaction between the decarboxylase mimic pep- tides and oxaloacetate was studied by UV spectroscop . A rate enhancement of a factor of approximately 40 over that of the butylamine catalysed reaction was observed. The overall reaction catalysed by the transaminase mimetic peptide 4 and the decarboxylase mimetic peptide 6 was directly followed by 1H NMR spectroscopy. The result is shown in figure lb as a section of the 1H NMR spectrum, at an early stage (foreground) and at a late stage (background) , of the transamination and the decarboxylation reactions catalysed by peptide 4 and peptide 6, re- spectively. At 3.66 ppm the oxaloacetate is shown and at 2.36 ppm the resonance of pyruvate is clearly visible. The pyruvate formed in the decarboxylation reaction is aminated by the pyridoxamine phosphate produced in the transamination reaction and forms alanine. It is evident that the catalysts are capable of catalysing the complete reaction sequence. The pyruvate formed in the decarboxylation reaction is then aminated by the pyridoxamine phosphate produced in the transamination reaction and forms alanine. It was found that more than 5 equivalents of alanine was formed per 1 equivalent of peptide. The catalyst according to the invention is thus capable of many turnovers. The maximum number of turnovers per catalytic peptide may not be limited to 5, even though this was used in the example. Example 4 The efficiency of peptide 4 in transammation under conditions of a 20-fold excess of ammo acid over peptide catalyst was compared to a reference catalyst, comprising 3 Arg, 1 Lys and 1 His, but having a configuration different from peptide 4. The sequence of the reference catalyst was : Ac-Asn-Ala-Ala-Asp-Nle-Glu-Gln-Ala-Ile-Lys-Gln-Leu-Ala- Glu-His-Nle-Ala-Ala-Gln-Gly-Pro-Val-Asp-Ala-Ala-Gln-Nle- Ala-Arg-Gln- eu-Ala-Arg-Glu-Phe-Glu-Ala-Phe-Ala-Arg-Ala- Gly It was found that when the transammation reaction using the naturally occurring form of aspartic acid, L- Asp was catalysed by peptide 4 or by the reference catalyst the reaction rate, measured as the initial rate, was more than 1000 times larger with the catalyst according to the invention than with the reference catalyst, al- though both peptides were at the same concentration. When the non-natural form of aspartic acid, D-Asp, was used, the transammation was a further 3 times faster than with L-Asp and thus more than 3000 times faster that the reaction of L-Asp catalysed by the reference peptide. It is thus clear that the catalyst according to the invention is capable of discrimination between enatiomeπc substrates, which is further illustrated m example 7. Example 5 In this example the reactivity of the catalytic peptides according to the invention was studied. The peptide according to the invention used was peptide 4, and it was used to catalyse transamination of aspartic acid, leading to formation of oxaloacetate. The reaction mechanism for this transamination is shown in figure 3. The reaction rates were measured by following the disappearance of pyrodixal phosphate at 390 nm and the appearance of pyridoxamine phosphate at 330 nm. The results are shown in the UV spectra in figure 4. The times indicated in the figure are the time passed after mixing 0.5 mM of peptide 4, 0.5 mM PPal (pyridoxal phosphate) , and 5 mM L-Asp in 50 mM Hepes at pH 7.4 and 25°C. Example 6 In this example two peptides according to the inven- tion, peptide 4 and peptide 6, were used as coupled catalysts in pyrodixal phosphate dependent transamination of L-Asp. It was found that the reactivity of the coupled catalysts (peptide 4 and peptide 6) is five times larger than that of the reactivity of only peptide 4. Peptide 6 is also reactive towards L-Asp, but slightly less than peptide 4. Since the reactivity of the two peptides combined is larger than the sum of the reactivities of each peptide reacting separately the heterodimer must be the reactive species and more reactive than each of the con- tributing peptides. The heterodimer therefore shows synergistic catalytic capacity. The results are shown in figures 6 A and 6 B. 0.5 mM of peptide 4, 0.5 mM of peptide 6, o.5 mM PPal, 5 mM L-Asp and 50 mM Hepes were used in the experiments at pH 7.4 and 25°C. A modelled struc- ture of the heterodimer is shown in figure 9. Example 7 In this example the enantioselectivity in transamination was studied. It was shown that the peptides ac- cording to the invention, in this case peptide 4, show differences in reactivity towards D- and L-amino acids, in this case D- and L-Ala, as illustrated by differences in initial rates, measured by absorbances at 390 n. The results are shown in figure 7. Similar results (not shown) were obtained for Lys and Nle. Comparative example A Peptides similar to the peptides according to the invention were also studied. It was found that the peptides with the following sequences show no measurable production of pyridoxamine phosphate within 20 minutes (1200 s) and are thus not reactive. These peptides contain arginines and histidines but no lysines. Comparative peptide I : Ac-Asn-Ala-Ala-Asp-Nle-Glu-His-Arg-Ile-Gln-Gln-Leu-Ala- Glu-His-Nle-Ala-Ala-Gln-Gly-Pro-Val-Asp-Ala-Ala-Gln-Nle- Ala-Glu-Gln-Leu-Ala-His-Arg-Phe-Glu-Ala-Phe-Ala-Arg-Ala- Gly-NH 2 Comparative peptide II: Ac-Asn-Ala-Ala-Asp-Nle-Glu-His-Arg-Ile-Gln-Gln-Leu-Ala- Glu-His-Nle-Ala-Ala-Gln-Gly-Pro-Val-Asp-Ala-Ala-Gln-Nle- Ala-Glu-Gln-Leu-Ala-His-Gin-Phe-Glu-Ala-Phe-Ala-Arg-Ala- Gly-NH 2 Comparative peptide III: Ac-Asn- la-Ala-Asp-Nle-Glu-His-Ala-lie-Gin-Arg-Leu-Ala- Glu-His-Nle-Ala-Ala-Arg-Gly-Pro-Val-His-Ala-Ala-Arg-Nle- Ala-Glu-Arg-Leu-Ala-Arg-His-Phe-Glu-Ala-Phe-Ala-Arg-Ala- Gly-NH 2 Comparative peptide IV: Ac-Asn-Ala-Ala-Asp-Nle-Glu-His-Ala-lie-Gin-Arg- eu-Ala- Glu-Arg-Nle-Ala-Ala-Arg-Gly-Pro-Val -His-Ala-Ala-Arg-Nle- Ala-Glu-His -Leu-Ala-Arg-His-Phe-Glu-Ala-Phe-Ala-Arg-Ala- Gly-NH 2 Comparative example B Finally another group of peptides similar to the peptides according to the invention was studied, but as in comparative example A, none of these peptides showed any measurable production of pyridoxamine phosphate within 20 minutes (1200 s) and they are thus not reactive. These peptides contain arginines, histidines and lysines. The peptides tested in this example had the following sequences : Comparative peptide V: Ac-Asn-Ala-Ala-Asp-Nle-Glu-His-Ala-lie-Gin-Lys-Leu-Ala- Glu-Arg-Nle-Ala-Ala-Arg-Gly-Pro-Val -His -Ala-Ala-Arg-Nle- Ala-Glu-Arg-Leu-Ala-Arg-His-Phe-Glu-Ala-Phe-Ala-Arg-Ala- Gly-NH 2 Comparative peptide VI: Ac -Asn-Ala-Ala-Asp-Nle -Glu-His -Ala - lie - Gin- Arg -Leu- Ala - Glu-Lys-Nle-Ala-Ala-Arg-Gly-Pro-Val -His -Ala-Ala-Arg-Nle- Ala-Glu-Arg-Leu-Ala-Arg-His -Phe -Glu-Ala- Phe -Ala -Arg-Ala- Gly-NH 2 Comparative peptide VII: Ac-Asn-Ala-Ala-Asp-Nle-Glu-His -Ala-lie-Gin-Arg-Leu-Ala- Glu-Arg-Nle-Ala-Ala-Arg-Gly- Pro-Val -His-Ala-Ala-Arg-Nle- Ala-Glu-Lys -Leu-Ala-Arg-His-Phe-Glu-Ala-Phe-Ala-Arg-Ala- Gly-NH 2

Claims: CLAIMS 1. A catalytic peptide consisting of a designed helix-loop-helix motif consisting of at least 37 amino ac- ids with lysine (Lys) , arginine (Arg) , histidine (His) , glutamic acid (Glu) , or aspartic acid (Asp) , independently of each other, in positions 8, 10, 11, 15, 19, 23, 26, 30, 34, and 37. 2. A catalytic peptide according to claim 1, con- sisting of 42 amino acids having leucine (Leu) in positions 12 and 31, isoleucine (lie) in position 9, phenylalanine (Phe) in positions 35 and 38, and norleucine (Nle) or leucin (Leu) in positions 5, 16 and 27. 3. A catalytic peptide according to claim 2, wherein said motif has SEQ. ID NO. 1 given in the appended sequence listing, Xaa in the sequence being Lys, Arg, His, Glu or Asp. 4. A catalytic peptide according to claim 2, wherein the amino acid in position 1 is acetylated at the N- terminal and amidated at the C-terminal. 5. A catalytic peptide according to claim 4, wherein said motif has SEQ. ID NO. 2 given in the appended sequence listing, Xaa in the sequence being Lys, Arg, His, Glu or Asp. 6. A catalytic peptide according to claim 3, wherein said peptide has SEQ. ID NO. 3 or SEQ. ID NO. 5 given in the appended sequence listing. 7. A catalytic peptide according to claim 5, wherein said peptide has SEQ. ID NO. 4 or SEQ. ID NO. 6 given in the appended sequence listing. 8. A catalytic molecule comprising the catalytic peptide according to claim 1. 9. A catalytic protein consisting of a naturally occurring protein wherein a naturally occurring sequence has been replaced by a catalytic peptide according to claim 1 with the same number of amino acids as in the naturally occurring sequence, wherein the amino acids in positions 1-7, 9, 12-14, 16-18, 20-22, 24-25, 27-29, 31- 33, 35-36, and 38 and above in the sequence replacing the naturally occurring sequence are the amino acids naturally occurring in the naturally occurring sequence, and wherein the amino acids in positions 8, 10, 11, 15, 19, 23, 26, 30, 34, and 37 retain the same relative positions they have in the catalytic peptide according to claim 1. 10. A catalytic protein according to claim 9, wherein said naturally occurring sequence is a helix- loop-helix motif. 11. A catalytic protein consisting of a naturally occurring protein to which a catalytic peptide according to any one of the claims 1-7 has been fused. 12. A catalytic protein consisting of a naturally occurring protein wherein a naturally occurring helix- loop-helix motif has been replaced by a catalytic peptide according to any one of the claims 1-7. 13. Use of a catalytic peptide according to any one of the claims 1-7, a catalytic molecule according to claim 8, or a catalytic protein according to any one of the claims 9-12 in combination with pyridoxal phosphate. 14. A catalyst for amino acid synthesis consisting essentially of two dimerised catalytic peptides according to any one of the claims 1-7 and pyridoxal phosphate. 15. A catalyst for amino acid synthesis consisting of a polypeptide or protein in which two dimerised catalytic peptides according to any one of the claims 1-7 and pyridoxal phosphate have been incorporated. 16. A catalyst according to claim 15, wherein said protein consists of a naturally occurring protein to which a catalytic peptide according to any one of the claims 1-7 has been fused. 17. A catalyst according to claim 15, wherein said protein consists of a naturally occurring protein wherein a naturally occurring helix-loop-helix motif has been replaced by a catalytic peptide according to any one of the claims 1-7. 18. A catalyst according to any one of the claims -17, for catalysis of a transamination reaction. 19. A catalyst according to any one of the claims -18, for catalysis of a decarboxylation reaction.

Inventors: BALTZER LARS, BALTZER, LARS
Applicants: A & SCIENCE INVEST AB, BALTZER LARS, A+ SCIENCE INVEST AB, BALTZER, LARS
Application year: 1999
Main classification: C07K14
IPC codes: C12N9