ENGINEERED METHIONINE GAMMA LYASE VARIANTS
The present invention provides engineered methionine gamma lyase polypeptides and compositions thereof. The engineered methionine gamma lyase polypeptides have been optimized to provide improved thermostability, protease stability, and stability under a range of pH conditions, including acidic (pH<7) conditions. The present invention also relates to the use of the compositions comprising the engineered methionine gamma lyase polypeptides for therapeutic purposes.
1 - 58 . (canceled)
59 . A method for treating and/or preventing the symptoms of homocystinuria in a subject, comprising administering to a subject having homocystinuria an effective amount of a recombinant methionine gamma lyase comprising a polypeptide sequence comprising at least 70% sequence identity to a reference sequence of SEQ ID NO: 2, 94, 248, 352, 478, 832, 916, 1172, 1232, 1346, or 1488, wherein the polypeptide sequence comprises one or more substitutions relative to the reference sequence of SEQ ID NO: 2, 94, 248, 352, 478, 832, 916, 1172, 1232, 1346, or 1488.
60 . The method of claim 59 , wherein administering the effective amount of the recombinant methionine gamma lyase results in reduction of plasma methionine and/or homocysteine by greater than 5%.
61 . The method of claim 59 , wherein the symptoms of homocystinuria are ameliorated.
62 . The method of claim 59 , wherein the subject is able to eat a diet that is less restricted in its methionine content or less restricted in its protein content than diets required by subjects exhibiting the symptoms of homocystinuria.
63 - 67 . (canceled)
68 . The method of claim 59 , wherein the recombinant methionine gamma lyase is administered immediately before, concurrently with, or immediately following ingestion of a protein meal containing methionine.
69 . (canceled)
70 . The method according to claim 59 , wherein the polypeptide sequence of the recombinant methionine gamma lyase comprises at least 70%, sequence identity to the reference sequence of SEQ ID NO: 2, wherein the polypeptide sequence comprises one or more substitutions relative to the reference sequence of SEQ ID NO: 2.
71 . The method of claim 70 , wherein the polypeptide sequence of the recombinant methionine gamma lyase comprises at least one or more substitutions at amino acid position: 2, 3, 4, 5, 6, 7, 8, 9, 11, 17, 21, 23, 25, 27, 29, 30, 31, 32, 33, 34, 35, 36, 38, 41, 43, 46, 47, 47, 48, 49, 50, 51, 53, 54, 55, 57, 58, 60, 62, 63, 66, 67, 68, 69, 82, 83, 87, 91, 99, 102, 111, 112, 113, 119, 124, 126, 127, 128, 132, 134, 138, 140, 141, 142, 144, 145, 146, 149, 150, 152, 154, 155, 156, 158, 160, 165, 167, 170, 173, 174, 175, 176, 177, 178, 179, 180, 181, 183, 189, 190, 193, 195, 199, 199, 206, 209, 214, 219, 220, 221, 223, 225, 228, 229, 230, 231, 232, 233, 236, 237, 239, 240, 242, 243, 245, 247, 250, 251, 252, 253, 254, 255, 256, 259, 263, 267, 269, 270, 271, 272, 275, 276, 278, 279, 281, 282, 283, 284, 287, 288, 290, 295, 296, 298, 300, 301, 304, 308, 309, 312, 314, 315, 316, 317, 322, 323, 324, 325, 327, 333, 334, 335, 336, 338, 341, 344, 348, 353, 357, 358, 361, 362, 364, 365, 366, 367, 368, 383, 384, 386, 388, 390, 391, 392, 394, 395, 396, 398, or combinations thereof, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 2.
72 . The method of claim 70 , wherein the polypeptide sequence of the recombinant methionine gamma lyase comprises at least one or more substitutions at amino acid position: 8, 11, 23, 55, 57, 69, 141, 165, 173, 189, 219, 223, 236, 237, 263, 267, 276, 279, 283, 290, 298, 304, 334, 341, 344, 366, 368, or combinations thereof, wherein the amino acid positions are relative to the reference of SEQ ID NO: 2.
73 . The method of claim 72 , wherein the polypeptide sequence of the recombinant methionine gamma lyase comprises one or more of the following substitutions: 8S, 11M, 23K, 55K/S, 57Y, 69I, 141E, 165R, 173V, 189L, 219VL, 2231, 236R, 237K, 263G, 267E, 276I, 279F, 283H, 290A/Q, 298S, 304R, 334L, 341F, 344A, 366E, 368D, or combinations thereof, wherein the amino acid positions relative to the reference sequence of SEQ ID NO: 2.
74 . The method of claim 70 , wherein the polypeptide sequence of the recombinant methionine gamma lyase comprises at least one substitution or substitution set at position 2, 6/25/1938/141/173/275/366/395, 6/25,1955, 6/38/55/138, 6/38/55/138/141/288/308, 6/38/55/141/180/275/362, 6/38/55/141/180/288/362/366, 6/38/55/275/288/308, 6/38/138/141/173/288/366, 6/38/141/288/366, 6/55/138/173/366, 6/55/283/362/366, 6/180/362/366, 25/38/55/275/288, 25/180/288/362, 38/55/180/362/366, 38/55/362, 38/141/173/288/308/366, 38/141/308/362/366, 38/173, 54, 54/145, 55, 69, 69/138/140/189/199/336, 69/138/145/189/199/290/344, 69/138/165/189/290/296/322/336/344/398, 69/138/189/199/366, 69/138/189/336/344, 69/138/199/263/322/344/366, 69/138/336/366, 69/140/145/322/348, 69/140/165/189/322/366, 69/145/165/296/336/344/366, 69/145/165/322, 69/145/189/199/290, 69/145/189/366/398, 69/145/199/336/344, 69/145/322/344, 69/145/344, 69/165/189/199/263/336/366, 69/165/189/263/322/336, 69/165/263/290/336, 69/165/296/398, 69/165/322, 69/165/322/344/366, 69/165/344/366, 69/189/290/344/366, 69/189/322, 69/290/344, 69/322/344/398, 138/189/263/322/366, 140/145/189, 140/145/189/322/344/366, 140/165/296/322/336, 142, 145/189, 145/189/199/263/296/336/344/366, 145/189/199/322/344/398, 145/263/290/344/398, 145/290, 152, 165/189/199/322/366, 165/322/336, 165/336/344, 170, 173/366, 177, 179, 179/251, 189, 189/290/322/336/366, 189/290/322/344, 189/290/366, 189/322, 189/322/344, 193, 232, 267, 271, 275, 278, 287, 295, 296/344, 301, 304, 309, 322, 327, 333, 361, 366, 392, and 395, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 2.
75 . The method of claim 70 , wherein the polypeptide sequence of the recombinant methionine gamma lyase comprises at least one substitution or substitution set at positions:
8/11/2023/55/57/69/141/165/173/189/219/223/233G/236/237/263/267/276/279/283/290/29 8/304/341/366/368/395,
8/11,2023/55/57/69/141/165/173/189/219/223/233/236/237/263/267/276/279/283/290/298/304/341/362/366/368,
8/11/2023/55/57/69/141/165/173/189/219/223/236/237/263/267/276/279/283/290/298/304/341/362/366/368/384,
8/11/2023/55/57/69/141/165/173/189/219/223/236/237/263/267/276/279/283/290/298/304/341/366/368/398,
8/11/2023/55/57/69/141/165/173/189/219/223/236/237/263/267/276/279/283/290/298R/30 4/341/366/368,
8/11/2023/55/57/69/141/165/173/189/219/223/230/233/236/237/263/267/276/279/283/290/298/304/341/366/368/384/395/398,
8/11/2023/55/57/69/141/165/173/189/219/223/230/236/237/263/267/276/279/283/290/298/304/341/362/366/368/395,
8/11/2023/55/57/69/141/165/173/189/219/223/230/233/236/237/263/267/276/279/283/290/298/304/308/341/366/368/384/395,
8/11/2023/55/57/69/141/165/173/189/219/223/230/233/236/237/263/267/276/279/283/290/298/301/304/341/366/368/395,
8/11/2023/55/57/69/141/165/173/189/219/223/230/233/236/237/263/267/276/279/283/290/298/304/341/366/368/384/395,
8/11/2023/55/57/69/141/165/173/189/219/223/233/236/237/263/267/276/279/283/290/298/304/341/366/368/384/395,
8/11/2023/55/57/69/141/165/173/189/219/223/233/236/237/263/267/276/279/283/290/298/304/341/362/366/368/395/398,
8/11/2023/55/57/69/141/165/173/189/219/223/230/236/237/263/267/276/279/283/290/298/304/341/366/368,
8/11/2023/55/57/69/141/165/173/189/219/223/230/233/236/237/263/267/276/279/281/283/290/298/304/341/366/368,
8/11/2023/55/57/69/141/165/173/189/219/223/230/233/236/237/263/267/276/279/283/290/298/304/341/366/368/395/398,
8/11/2023/55/57/69/141/165/173/189/219/223/230/233/236/237/263/267/276/279/283/290/298/304/341/362/366/3,
8/11/2023/55/57/69/141/165/173/189/219/223/230/233/236/237/263/267/276/279/283/290/298/304/341/362/366/368/395,
8/11/2023/55/57/69/141/165/173/189/219/223/233/236/237/263/267/276/279/283/290/298/304/308/341/366/368/384/395,
8/11/2023/55/57/69/141/165/173/189/219/223/233/236/237/263/267/276/279/283/290/298/304/341/362/366/368/395,
8/11/2023/55/57/69/141/165/173/189/219/223/236/237/263/267/276/279/283/290/298/301/304/341/366/368,
8/11/2023/55/57/69/141/165/173/189/219/223/233/236/237/263/267/276/279/283/290/298/304/341/366/368,
8/11/2023/55/57/69/141/165/173/189/219/223/236/237/263/267/276/279/283/290/298/304/341/366/368/384/398,
8/11/2023/55/57/69/141/165/173/189/219/223/236/237/263/267/276/279/283/290/298/304/341/366/368/395/398,
8/11/2023/55/57/69/141/165/173/189/219/223/236/237/263/267/276/279/283/290/298/301S/304/308/341/366/368/384,
8/11/2023/55/57/69/141/165/173/189/219/223/233/236/237/263/267/276/279/283/290/298/304/341/362/366/368,
8/11/2023/55/57/69/141/165/173/189/219/223/230/233/236/237/263/267/276/279/283/290/298/301/304/341/362/366/368/384/395,
8/11/2023/55/57/69/141/165/173/189/219/223/236/237/263/267/276/279/283/290/298/304/341/362/366/368/384,
8/11/2023/55/57/69/141/165/173/189/219/223/233/236/237/263/267/276/279/283/290/298/304/341/366/368,
8/11/2023/55/57/69/141/165/173/189/219/223/233/236/237/263/267/276/279/283/290/298/301/304/341/362/366/368, or
8/11/2023/55/57/69/141/165/173/189/219/223/230/233/236/237/263/267/276/279/283/290/298/301/304/341/366/368/395,
wherein the amino acid positions of the polypeptide sequence are relative to the reference sequence of SEQ ID NO: 2.
76 . The method of claim 70 , wherein the polypeptide sequence of the recombinant methionine gamma lyase comprises at least one substitution or substitution set:
8S/11M/23K/55S/57Y/69I/141E/165R/173V/189L/219L/223I/233S/236R/237K/263G/26 7E/276I/279F/283H/290Q/298S/304R/341F/362E/366E/368D,
8S/11M/23K/55S/57Y/69I/141E/165R/173V/189L/219L/223I/233M/236R/237K/263G/2 67E/276I/279F/283H/290Q/298N/304R/341F/362E/366E/368D,
8S/11M/23K/55S/57Y/69I/141E/165R/173V/189L/219L/223I/236R/237K/263G/267E/2 761/279F/283H/290Q/298N/304R/341F/362E/366E/368D/384G, 8S/11M/23K/55S/57Y/69I/141E/165R/173V/189L/219L/223I/236R/237K/263G/267E/2 761/279F/283H/290Q/298S/304R/341F/366E/368D/398G, 8S/11M/23K/55S/57Y/69I/141E/165R/173V/189L/219L/223I/236R/237K/263G/267E/2 761/279F/283H/290Q/298R/304R/341F/366E/368D,
8S/11M/23K/55S/57Y/69I/141E/165R/173V/189L/219L/223I/230V/233Q/236R/237K/2 63G/267E/276I/279F/283H/290Q/298S/304R/341F/366E/368D/384G/395R/398G,
8S/11M/23K/55S/57Y/69/141E/165R/173V/189L/219L/223I/230V/236R/237K/263G/2 67E/276I/279F/283H/290Q/298S/304R/341F/362E/366E/368D/395R,
8S/11M/23K/55S/57Y/69I/141E/165R/173V/189L/219L/223I/230V/233Q/236R/237K/2 63G/267E/276I/279F/283H/290Q/298S/304R/308K/341F/366E/368D/384G/395R,
8S/11M/23K/55S/57Y/69I/141E/165R/173V/189L/219L/223I/230V/233M/236R/237K/2 63G/267E/276I/279F/283H/290Q/298N/301S/304R/341F/366E/368D/395R,
8S/11M/23K/55S/57Y/69I/141E/165R/173V/189L/219L/223I/230V/233M/236R/237K/2 63G/267E/276I/279F/283H/290Q/298S/304R/341F/366E/368D/384G/395R,
8S/11M/23K/55S/57Y/69I/141E/165R/173V/189L/219L/223I/233Q/236R/237K/263G/2 67E/276I/279F/283H/290Q/298S/304R/341F/366E/368D/384G/395R,
8S/11M/23K/55S/57Y/69I/141E/165R/173V/189L/219L/223I/233S/236R/237K/263G/26 7E/276I/279F/283H/290Q/298S/304R/341F/366E/368D/384G/395R,
8S/11M/23K/55S/57Y/69I/141E/165R/173V/189L/219L/223I/233M/236R/237K/263G/2 67E/276I/279F/283H/290Q/298S/304R/341F/362E/366E/368D/395R/398G,
8S/11M/23K/55S/57Y/69I/141E/165R/173V/189L/219L/223I/230V/236R/237K/263G/2 67E/276I/279F/283H/290Q/298S/304R/341F/366E/368D,
8S/11M/23K/55S/57Y/69I/141E/165R/173V/189L/219L/223I/230V/233Q/236R/237K/2 63G/267E/276I/279F/281V/283H/290Q/298S/304R/341F/366E/368D,
8S/11M/23K/55S/57Y/69I/141E/165R/173V/189M/219L/223I/230V/233S/236R/237K/2 63G/267E/276I/279F/283H/290Q/298N/304R/341F/366E/368D/395R/398G, 8S/11M/23K/55S/57Y/69I/141E/165R/173V/189L/219L/223I/230V/233Q/236R/237K/2 63G/267E/276I/279F/283H/290Q/298N/304R/341F/362E/366E/368D,
8S/11M/23K/55S/57Y/69I/141E/165R/173V/189L/219L/223I/230V/233M/236R/237K/2 63G/267E/276I/279F/283H/290Q/298S/304R/341F/362E/366E/368D/395R,
8S/11M/23K/55S/57Y/69I/141E/165R/173V/189L/219L/223I/233M/236R/237K/263G/2 67E/276I/279F/283H/290Q/298N/304R/308K/341F/366E/368D/384G/395R,
8S/11M/23K/55S/57Y/69I/141E/165R/173V/189L/219L/223I/233S/236R/237K/263G/26 7E/276I/279F/283H/290Q/298S/304R/341F/362E/366E/368D/395R,
8S/11M/23K/55S/57Y/69I/141E/165R/173V/189L/219L/223I/236R/237K/263G/267E/2 761/279F/283H/290Q/298Y/301S/304R/341F/366E/368D,
8S/11M/23K/55S/57Y/69I/141E/165R/173V/189L/219L/223I/233M/236R/237K/263G/2 67E/276I/279F/283H/290Q/298S/304R/341F/366E/368D,
8S/11M/23K/55S/57Y/69I/141E/165R/173V/189L/219L/223I/236R/237K/263G/267E/2 761/279F/283H/290Q/298S/304R/341F/366E/368D/384G/398G,
8S/11M/23K/55S/57Y/69I/141E/165R/173V/189L/219L/223I/236R/237K/263G/267E/2 761/279F/283H/290Q/298S/304R/341F/366E/368D/395R/398G,
8S/11M/23K/55S/57Y/69I/141E/165R/173V/189L/219L/223I/236R/237K/263G/267E/2 761/279F/283H/290Q/298N/301S/304R/308K/341F/366E/368D/384G,
8S/11M/23K/55S/57Y/69I/141E/165R/173V/189L/219L/223I/233Q/236R/237K/263G/2 67E/276I/279F/283H/290Q/298R/304R/341F/362E/366E/368D,
8S/11M/23K/55S/57Y/69I/141E/165R/173V/189L/219L/223I/230V/233Q/236R/237K/2 63G/267E/276I/279F/283H/290Q/298R/301S/304R/341F/362E/366E/368D/384G/395R,
8S/11M/23K/55S/57Y/69/141E/165R/173V/189L/219L/223I/230V/233S/236R/237K/26 3G/267E/276I/279F/283H/290Q/298N/301S/304R/341F/362E/366E/368D/384G/395R,
8S/11M/23K/55S/57Y/69I/141E/165R/173V/189L/219L/223I/236R/237K/263G/267E/2 761/279F/283H/290Q/298S/304R/341F/362E/366E/368D/384G,
8S/11M/23K/55S/57Y/69I/141E/165R/173V/189L/219L/223I/233Q/236R/237K/263G/2 67E/276I/279F/283H/290Q/298S/304R/341F/366E/368D,
8S/11M/23K/55S/57Y/69I/141E/165R/173V/189L/219L/223I/233G/236R/237K/263G/2 67E/276I/279F/283H/290Q/298S/304R/341F/366E/368D/395R,
8S/11M/23K/55S/57Y/69I/141E/165R/173V/189L/219L/223I/233Q/236R/237K/263G/2 67E/276I/279F/283H/290Q/298N/301S/304R/341F/362E/366E/368D,
8S/11M/23K/55S/57Y/69I/141E/165R/173V/189L/219L/223I/230V/233Q/236R/237K/2 63G/267E/276I/279F/283H/290Q/298R/301S/304R/341F/366E/368D/395R, or
8S/11M/23K/55S/57Y/69I/141E/165R/173V/189L/219L/223I/230V/233Q/236R/237K/2 63G/267E/276I/279F/283H/290Q/298N/301S/304R/341F/366E/368D/395R,
wherein the amino acid positions of the polypeptide sequence are relative to the reference sequence of SEQ ID NO: 2.
77 . The method of claim 59 , wherein the polypeptide sequence of the recombinant methionine gamma lyase comprises at least 80% sequence identity to the reference sequence of SEQ ID NO: 94, 248, 352, 478, 832, 916, 1172, 1232, 1346, or 1488.
78 . The method of claim 77 , wherein the polypeptide sequence of the recombinant methionine gamma lyase comprises at least one or more substitutions at amino acid position: 2, 3, 4, 5, 6, 7, 8, 9, 11, 17, 21, 23, 25, 27, 29, 30, 31, 32, 33, 34, 35, 36, 38, 41, 43, 46, 47, 47, 48, 49, 50, 51, 53, 54, 55, 57, 58, 60, 62, 63, 66, 67, 68, 69, 82, 83, 87, 91, 99, 102, 111, 112, 113, 119, 124, 126, 127, 128, 132, 134, 138, 140, 141, 142, 144, 145, 146, 149, 150, 152, 154, 155, 156, 158, 160, 165, 167, 170, 173, 174, 175, 176, 177, 178, 179, 180, 181, 183, 189, 190, 193, 195, 199, 199, 206, 209, 214, 219, 220, 221, 223, 225, 228, 229, 230, 231, 232, 233, 236, 237, 239, 240, 242, 243, 245, 247, 250, 251, 252, 253, 254, 255, 256, 259, 263, 267, 269, 270, 271, 272, 275, 276, 278, 279, 281, 282, 283, 284, 287, 288, 290, 295, 296, 298, 300, 301, 304, 308, 309, 312, 314, 315, 316, 317, 322, 323, 324, 325, 327, 333, 334, 335, 336, 338, 341, 344, 348, 353, 357, 358, 361, 362, 364, 365, 366, 367, 368, 383, 384, 386, 388, 390, 391, 392, 394, 395, 396, 398, or combinations thereof, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 94, 248, 352, 478, 832, 916, 1172, 1232, 1346, or 1488.
79 . The method of claim 77 , wherein the polypeptide sequence of the recombinant methionine gamma lyase comprises one or more of the following amino acid residues: 2K/S/T/W, 3A/E/P/S, 4E/G/R/V, 5G/H/I/K/P/T, 6L/P/R/S, 7E/F/G/L/S/V, 8G/P/R/S/Y, 9A/R, 11S/A/C/G/H/M/R, 17C, 21A, 23S/K, 25E, 27S, 29T, 30N, 31A, 32N, 33A/T, 34L, 35G/S, 36S, 38F, 41E, 43A, 46Q, 47G, 47Q, 48M/R, 49A/T, 50L/M/V, 51K/N/S, 53L, 54I/Q/R, 55E/H/K/P/A/N, 57Y/H, 58T, 60T, 62A/C/H, 63G, 66Q/S, 67A/Q/R/V, 68D/G, 69R/V/I/W, 82C, 83S, 87G, 91S/L, 99A, 102A, 111H, 112A, 113M/T, 119T, 124A/H/R/S/V, 126A/E/R/S, 127A/K/Q/V, 128H, 132V, 134F, 138C/S/T, 140T, 141Q/V/E/G/P/R/S/T, 142L/S, 144R, 145G/S, 146S, 149D/T, 150T, 152A, 154V, 155F, 156L, 158S, 160M, 165R, 167T, 170G/P/Q/W, 173V/A, 174E, 175V, 176V, 177T, 178A, 179A/S/W, 180G/V, 181I, 183V, 189I/M/P/S/Y/L, 190A/G/L, 193S, 195S/T, 199T, 199W, 2061, 209G, 214C, 219L/V/I, 220I/V, 221G, 223L/I, 225C, 228A/R, 229G/H/S, 230V, 231V, 232P, 233G/M/Q/S/V, 236A/C/R/Y, 237A/G/H/K/L/R/T/V/Y, 239C/K/T/V/Y, 240D/G/P/R, 242F, 243C/G/S, 245G/S, 247M, 250F/S, 251A/N, 252C, 253G/M, 254A, 255V, 256L/V, 259L, 263G/K/P/Q/S/A, 267N/E/R/T, 269S, 270S, 271D, 272S, 275A/H/N/R/S, 276V/I/L, 278C, 279H/F/W, 281V, 282A/N/Q/V, 283N/Q/H, 284A/E/S/V, 287D/V, 288A/G/I/K/M/Q/R/S/T/W, 290N/Q/V/A/K, 295G/S/E, 296G/N, 298G/K/P/S/V/C/G/N/R/Y, 300R, 301D/E/G/K/N/Q/S, 304R/K/V, 308A/K/L/S, 309A/K, 312A, 314L, 315S, 316L, 317R, 322A/E/K/Q/V, 323K/S/T, 324S, 325A, 327M, 333F/S/T, 334V/L, 3351, 336H, 338G, 341F, 344C/T/V/A/G/R/S, 348V, 353T, 357A/G, 358C/K/L/R/T, 361V, 362R/E, 364L, 365K/R, 366Q/R/E, 367A, 368G/D, 3831, 384A/G/Q/S, 386I, 388R, 390L, 391N/E, 392H, 394P, 395A/D/H/R, 396Q/R/V, 398G/P/Q, or combinations thereof, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 94, 248, 352, 478, 832, 916, 1172, 1232, 1346, or 1488.
80 . The method of claim 77 , wherein the polypeptide sequence of the recombinant methionine gamma lyase comprises at least one or more substitutions at amino acid position: 8, 11, 23, 55, 57, 69, 141, 165, 173, 189, 219, 223, 236, 237, 263, 267, 276, 279, 283, 290, 298, 304, 334, 341, 344, 366, 368, or combinations thereof, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 94, 248, 352, 478, 832, 916, 1172, 1232, 1346, or 1488.
81 . The method of claim 77 , wherein the polypeptide sequence of the recombinant methionine gamma lyase comprises one or more of the following amino acid residues: 8S, 11M, 23K, 55K/S, 57Y, 69I, 141E, 165R, 173V, 189L, 219V/L, 2231, 236R, 237K, 263G, 267E, 276I, 279F, 283H, 290A/Q, 298S, 304R, 334L, 341F, 344A, 366E, 368D, or combinations thereof, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 94, 248, 352, 478, 832, 916, 1172, 1232, 1346, or 1488.
82 . The method of claim 59 , wherein the polypeptide sequence of the recombinant methionine gamma lyase comprises at least 80% sequence identity to SEQ ID NO: 1488, wherein the polypeptide sequence comprises at least one substitution or substitution set at one or more positions 6, 9, 63, 69, 82, 93, 126, 141, 155, 175, 189, 199, 220, 221, 229, 230, 233, 253, 254, 282, 284, 298, 300, 301, 304, 308, 323, 362, 365, 384, 395, 396, and 398, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 1488.
83 . The method of claim 59 , wherein the polypeptide sequence of the recombinant methionine gamma lyase comprises at least 80% sequence identity to SEQ ID NO: 1488, wherein the polypeptide sequence comprises at least one substitution or substitution set at one or more positions 189/230/233/298/395/398, 230, 230/233/281, 230/233/298/301/362/384/395, 230/233/298/301/395, 230/233/298/362, 230/233/308/384/395, 230/233/362/395, 230/233/384/395, 230/233/384/395/398, 230/362/395, 233, 233/298/301/362, 233/298/308/384/395, 233/298/362, 233/362, 233/362/395, 233/362/395/398, 233/384/395, 233/395, 298, 298/301, 298/301/308/384, 298/362/384, 362/384, 384/398, 395/398, and 398, wherein the amino acid positions are relative to the reference sequence of SEQ ID NO: 1488.
84 . The method of claim 59 , wherein the polypeptide sequence of the recombinant methionine gamma lyase comprises an even-numbered sequence of SEQ ID NOS: 4-1734.
85 . The method of claim 59 , wherein the polypeptide sequence of the recombinant methionine gamma lyase comprises the polypeptide sequence of SEQ ID NO: 94, 248, 352, 478, 832, 916, 1172, 1232, 1346, 1488 or 1706.
86 . The method of claim 59 , wherein the polypeptide sequence of the recombinant methionine gamma lyase comprises at least 80% sequence identity to SEQ ID NO: 2, wherein the polypeptide comprises the following substitutions relative to the reference sequence of SEQ ID NO: 2: T8S, S11M, S23K, G55S, H57Y, L69I, Q141E, Q165R, A173V, Y189L, 1219L, L223I, R233S, L236R, E237K, A263G, D267E, V276I, H279F, Q283H, N290Q, A298S, Q304R, L341F, Q362E, H366E, G368D, and K395R.
87 . The method of claim 59 , wherein the polypeptide sequence of the recombinant methionine gamma lyase comprises at least 80% sequence identity to SEQ ID NO: 1488, wherein the polypeptide comprises the following substitutions relative to the reference sequence of SEQ ID NO: 1488: R233S, Q362E, and K395R.
88 . The method of claim 59 , wherein the polypeptide sequence of the recombinant methionine gamma lyase comprises SEQ ID NO: 1706.