1. KtzJ-dependent serine activation and O-methylation by KtzH for kutznerides biosynthesis
Olga E Zolova, Sylvie Garneau-Tsodikova J Antibiot (Tokyo). 2014 Jan;67(1):59-64. doi: 10.1038/ja.2013.98. Epub 2013 Oct 9.
Kutznerides are hexadepsipeptide antifungal and antimicrobial agents containing O-methyl-L-serine in their very unique peptidic backbone. During kutznerides biosynthesis, this O-methylated amino-acid residue is proposed to result from the action of an adenylation (A) domain present in KtzH, which is interrupted by the S-adenosylmethionine-binding-containing part of a methyltransferase. In this study, we co-expressed recombinant KtzH(A4MA4T4) with its MbtH-like protein partner KtzJ and demonstrated the requirement for KtzJ in producing soluble and active KtzH(A4MA4T4). We demonstrated the specificity of KtzH(A4MA4T4) toward L-Ser and showed the activity of the partial methyltransferase enzyme in O-methylation of L-Ser after its covalent attachment to the thiolation domain of KtzH(A4MA4T4). The insights gained from this work may guide future study and development of engineered interrupted adenylation domains for combinatorial biosynthetic methodologies.
3. Purification and characterization of S-alkylcysteine alpha,beta-lyase from Pseudomonas putida
H Kamitani, N Esaki, H Tanaka, H Imahara, K Soda J Nutr Sci Vitaminol (Tokyo). 1990 Aug;36(4):339-47. doi: 10.3177/jnsv.36.4-supplementi_339.
S-Alkylcysteine alpha,beta-lyase [EC 4.4.1.6] was purified to more than 90% homogeneity from the cell extract of Pseudomonas putida ICR 3640. The enzyme has a molecular weight of about 195,000, and is composed of six subunits identical in molecular weight (37,000). Pyridoxal 5'-phosphate is required as a cofactor. The enzyme catalyzes the alpha,beta-elimination of S-methyl-L-cysteine and its analogs such as S-ethyl-L-cysteine, L-djenkolate, Se-methyl-DL-selenocysteine, and O-methyl-L-serine. However, S-methyl-D-cysteine, L-methionine, and L-norvaline were inert. The enzyme catalyzes also the beta-replacement reaction of the thiomethyl group of S-methyl-L-cysteine with various thiols to yield the corresponding S-substituted cysteines. In addition to S-methyl-L-cysteine, Se-methyl-DL-selenocysteine and O-methyl-L-serine also serve as substrates in the beta-replacement reaction.
