Variant polypeptides capable of aminating aliphatic alpha keto acids

Abstract

Disclosed are, among other things, variant polypeptides, nucleic acids encoding the polypeptides, production of the variant polypeptides, and use of the variant polypeptides in various applications, such as screening and synthetic methods. For example, the variant polypeptides, or enzymatically-active fragments thereof, are useful for converting aliphatic keto acids to aliphatic alpha amino acids.

1 - 10 . (canceled) 
     
     
         11 . A nucleic acid encoding a polypeptide;
 wherein the polypeptide comprises:   (a) the amino acid sequence of any one of SEQ ID NOS:2 and 13-18, wherein X is not leucine;   (b) an amino acid sequence that is at least 95% identical to: (i) amino acids 6 to 238 of SEQ ID NO:2; (ii) amino acids 7 to 237 of SEQ ID NO:13; (iii) amino acids 4 to 236 of SEQ ID NO:14; (iv) amino acids 4 to 236 of SEQ ID NO:15; (v) amino acids 4 to 236 of SEQ ID NO:16; (vi) amino acids 4 to 236 of SEQ ID NO:17; or (vii) amino acids 4 to 236 of SEQ ID NO:18, wherein X is not leucine;   (c) an amino acid sequence that is at least 95% identical to: (i) amino acids 6 to 298 of SEQ ID NO:2; (ii) amino acids 7 to 297 of SEQ ID NO:13; (iii) amino acids 4 to 296 of SEQ ID NO:14; (iv) amino acids 4 to 296 of SEQ ID NO:15; (v) amino acids 4 to 296 of SEQ ID NO:16; (vi) amino acids 4 to 296 of SEQ ID NO:17; or (vii) amino acids 4 to 296 of SEQ ID NO:18, wherein X is not leucine; or   (d) the amino acid sequence of SEQ ID NO: 4, 5, 6, or 20; and   X is isoleucine, valine, glycine, alanine, serine, or threonine.   
     
     
         12 . An expression vector, comprising the nucleic acid of  claim 11 . 
     
     
         13 . A cell, comprising the expression vector of  claim 12 . 
     
     
         14 . A method for producing a polypeptide, comprising culturing the cell of  claim 13  under conditions suitable for protein expression, thereby producing a polypeptide. 
     
     
         15 . The method of  claim 14 , further comprising isolating the polypeptide from the cell or from media in which the cell is cultured. 
     
     
         16 . (canceled) 
     
     
         17 . The nucleic acid of  claim 11 , wherein X is isoleucine. 
     
     
         18 . The nucleic acid of  claim 11 , wherein X is valine. 
     
     
         19 . The nucleic acid of  claim 11 , wherein X is glycine. 
     
     
         20 . The nucleic acid of  claim 11 , wherein X is alanine. 
     
     
         21 . The nucleic acid of  claim 11 , wherein X is serine or threonine. 
     
     
         22 . The nucleic acid of  claim 11 , wherein the amino acid sequence is SEQ ID NO:2. 
     
     
         23 . The nucleic acid of  claim 22 , wherein X is alanine. 
     
     
         24 . The expression vector of  claim 12 , wherein the expression vector is a prokaryotic expression vector. 
     
     
         25 . The expression vector of  claim 12 , wherein the expression vector is a eukaryotic expression vector. 
     
     
         26 . The cell of  claim 13 , wherein the cell is a bacterial cell. 
     
     
         27 . The cell of  claim 13 , wherein the cell is a mammalian cell. 
     
     
         28 . The cell of  claim 13 , wherein the cell is a yeast cell. 
     
     
         29 . The cell of  claim 13 , wherein the cell is an insect cell. 
     
     
         30 . The cell of  claim 13 , wherein the cell is a plant cell. 
     
     
         31 . The method of  claim 14 , wherein the polypeptide comprises:
 (a) the amino acid sequence of any one of SEQ ID NOS:2 and 13-18, wherein X is not leucine;   (b) an amino acid sequence that is at least 95% identical to: (i) amino acids 6 to 238 of SEQ ID NO:2; (ii) amino acids 7 to 237 of SEQ ID NO:13; (iii) amino acids 4 to 236 of SEQ ID NO:14; (iv) amino acids 4 to 236 of SEQ ID NO:15; (v) amino acids 4 to 236 of SEQ ID NO:16; (vi) amino acids 4 to 236 of SEQ ID NO:17; or (vii) amino acids 4 to 236 of SEQ ID NO:18, wherein X is not leucine;   (c) an amino acid sequence that is at least 95% identical to: (i) amino acids 6 to 298 of SEQ ID NO:2; (ii) amino acids 7 to 297 of SEQ ID NO:13; (iii) amino acids 4 to 296 of SEQ ID NO:14; (iv) amino acids 4 to 296 of SEQ ID NO:15; (v) amino acids 4 to 296 of SEQ ID NO:16; (vi) amino acids 4 to 296 of SEQ ID NO:17; or (vii) amino acids 4 to 296 of SEQ ID NO:18, wherein X is not leucine; or   (d) the amino acid sequence of SEQ ID NO: 4, 5, 6, or 20; and   X is isoleucine, valine, glycine, alanine, serine, or threonine.