嗜热玫瑰红球菌肌氨酸氧化酶(thermophilus sarcosine oxidase,TrSOX)是一种N-甲基脱除酶,对底物具有特异的手性选择性并且对高温和有机溶剂具有良好的耐受性。在同源家族中,Arg54是催化中心严格保守的5个残基之一,为了研究该位点对酶学性质的影响并筛选出具有良好催化效率的突变体,在预先构建的木糖诱导质粒pMA5-Pxyl-trsox中进行Arg54位点的定点饱和突变,并将得到的质粒转化到枯草芽孢杆菌WB600中进行表达。在19个突变体中,共有15个突变体可以表达并进行制备,其中几乎所有突变体的粗酶液蛋白含量相较野生型TrSOX都有所降低,只有R54D提高约2.5倍。所有突变体都保持了优异的热稳定性,除R54Q、R54N、R54K、R54D和R54V外,其他大多数突变体的二级结构组成均受到一定影响。突变体对底物的手性特异性与野生型TrSOX相同,能特异性识别N-甲基-L-氨基酸类底物,并能将甲基脱除。R54D和R54K对N-甲基-L-氨基酸类底物的催化效率显著提高,而其他突变体的催化效率降低甚至完全失活。此外,包括R54P、R54K和R54I在内的一些突变体能够识别新的非氨基酸相关底物,如1,2,3,4-四氢异喹啉、1-甲基-1,2,3,4-四氢异喹啉、己内酰胺、2-甲基哌啶、哌啶和(3S)-(+)-3-(甲氨基)吡咯烷等,拓展了TrSOX的底物适应性。
Thermomicrobium roseum sarcosine oxidase (TrSOX) is an N-demethylase with specific substrate chiral selectivity, good resistance against high temperature and organic solvents. TrSOX catalyze the methyl removal reaction of sarcosine to produce glycine, with the cofactor flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN). In the food industry, sarcosine-based oxidation biosensors have been widely used for the determination of organic acids and glycerol in wine fermentation. Arg54 is one of the conserved residues in the catalytic center, in order to investigate the effects of this site on the enzymatic properties and to screen out mutants with good catalytic efficiency, site-saturation mutagenesis at ArgLys54 was carried out in a pre-constructed xylose-induced plasmid pMA5-Pxyl-trsox, and the resulted plasmids were transformed to Bacillus subtilis WB600 for expression. All 15 of the 19 mutants could be expressed and prepared except for R54S, R54C, R54H, R54T and R54W,because plasmids carrying the four mutant genes mentioned above are unable to express soluble and active enzyme molecules in the host cell. Determination of the protein concentration of the crude enzyme solution revealed an increase in total protein for R54D, R54K, R54Q, R54V and an approximately 2.5-fold increase for R54D. The analysis of the properties of mutant TrSOX and natural TrSOX showed that mutant TrSOX maintained the excellent thermal stability of natural TrSOX, however, the secondary structures of most mutants were affected except R54Q, R54N, R54K, R54D and R54V. The reason for the change in secondary structure of most mutants may be that the ArgLys54 site is more important for the maintenance of the overall structure of TrSOX and its mutation affects the overall folding of the protein structure. Although the secondary structure of most of the mutants was affected, the thermal stability was not greatly affected, because the regions where the secondary structure was altered did not affect the unchaining temperature of the mutants and did not make a difference to the energy used to disrupt the overall structure of the protein, resulting in the mutants continuing the excellent thermal stability of natural TrSOX. In addition, the chiral specificity of mutants for substrates were same as those of the wild TrSOX; The mutants could specifically recognize N-methyl-L-amino acids and could deplete the methyl group, but no catalytic effect on the D-conformation. A comparative study of the catalytic activity of these mutants revealed that the catalytic efficiency of R54D and R54K for N -methyl- L-amino acids was significantly increased, while the other mutants showed reduced efficiency or even complete inactivation. For the basic residue mutant R54K, the catalytic efficiency was increased by 3.17-fold, 5.99-fold, 9.12-fold, 7.61-fold, 1.00-fold and 2.07-fold for N-methyl-glycine, N-methyl-L-alanine,N-methyl-L-phenylalanine, N-methyl-L-leucine,N-methyl-L-tryptophan and N-methyl-L-aspartic acid respectively. The mutant R54D containing acidic residues also showed a significant increase in the catalytic efficiency of N-methyl-L-amino acids, with 1.79-fold, 5.57-fold, 17.92-fold, 9.00-fold and 1.43-fold increases in the catalytic efficiency of N-methyl-glycine, N-methyl-L-alanine, N-methyl-L-phenylalanine, N-methyl-leucine and N-methyl-L-tryptophan, respectively .The catalytic effect of mutant TrSOX on N-methyl-L-amino acid substrates lies in the fact that the basic or acidic side chain of the R54 residue can interact directly with the active side chain of the substrate, directing the entry of the substrate and the release of the product, thereby enhancing catalytic properties. In addition, for non-amino acid substrates, natural TrSOX was inactive against all substrates except L-homoproline, and mutants such as R54I,R54Q,R54N,R54G,R54K,R54P,R54D showed catalytic activity not present in wild-type TrSOX when substrated with alkaloids such as pyrrolidine ((3S)-(+)-3-(methylamino)pyrrolidine), piperidine (piperidine and 2-methylpiperidine), even larger structures (caprolactam) or more complex ones (1,2,3,4-tetrahydroisoquinoline and 1-methyl-1,2,3,4-tetrahydroisoquinoline) as substrates, catalytic activity was detected that was not present in wild-type TrSOX. This suggests that the basic guanidino side chain of R54 may reject these substrates, which in the above mutant can enter the active pocket and be converted to the relevant product. The catalytic mechanism of mutant TrSOX for non-amino acid substrates is similar to the original catalytic mechanism, but further studies (LC-MS/NMR) are needed to clarify the products. However, the catalytic efficiency of these non-amino acid bound substrates is much lower than that of N-methylamino acids and, in addition, the catalytic mechanism and products of the non-amino acids are not yet known. In our further work, there is a need to further research the catalytic ability for such substrates and to purify and characterize the products.
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