Mechanistic investigations of the dehydration reaction of lacticin 481 synthetase using site-directed mutagenesis

Abstract

Lantibiotic synthetases catalyze the dehydration of Ser and Thr residues in their peptide substrates to dehydroalanine (Dha) and dehydrobutyrine (Dhb), respectively, followed by the conjugate addition of Cys residues to the Dha and Dhb residues to generate the thioether cross-links lanthionine and methyllanthionine, respectively. In this study ten conserved residues were mutated in the dehydratase domain of the best characterized family member, lacticin 481 synthetase (LctM). Mutation of His244 and Tyr408 did not affect dehydration activity with the LctA substrate whereas mutation of Asn247, Glu261, and Glu446 considerably slowed down dehydration and resulted in incomplete conversion. Mutation of Lys159 slowed down both steps of the net dehydration: phosphorylation of Ser/Thr residues and the subsequent phosphate elimination step to form the dehydro amino acids. Mutation of Arg399 to Met or Leu resulted in mutants that had phosphorylation activity but displayed greatly decreased phosphate elimination activity. The Arg399Lys mutant retained both activities, however. Similarly, the Thr405Ala mutant phosphorylated the LctA substrate but had compromised elimination activity. Finally, mutation of Asp242 or Asp259 to Asn led to mutant enzymes that lacked detectable dehydration activity. Whereas the Asp242Asn mutant retained phosphate elimination activity, the Asp259Asn mutant was not able to eliminate phosphate from a phosphorylated substrate peptide. A model is presented that accounts for the observed phenotypes of these mutant enzymes.

Figure 1

A. Dehydration of serine and threonine residues results in the formation of dehydroalanine (Dha) or dehydrobutyrine (Dhb), respectively. Subsequent Michael type addition of cysteines onto the unsaturated amino acids results in (methyl)lanthionine formation. B. The biosynthesis of lacticin 481 (12). Following ribosomal synthesis, lacticin 481 synthetase (LctM) dehydrates serine and threonine residues in the propeptide region of the substrate peptide LctA and catalyzes (methyl)lanthionine formation. LctT then removes the unmodified leader peptide and secretes the final product (13).

Figure 2

Partial sequence alignment of the N-terminal region of selected LanM enzymes. Fully conserved residues are highlighted in yellow. For a complete sequence comparison, see reference (9).

Figure 3

MALDI-TOF mass spectra of substrates (dashed line) and dehydration assays after 15 min (solid line) containing His₆-LctA and (A) LctM-H244N, or (B) LctM-Y408F.

Figure 4

MALDI-TOF mass spectra of substrates (dashed line) and dehydration assays (solid line) after incubation of His₆-LctA with (A) LctM-E261Q for 1 h, (B) LctM-E446M for 6 h, (C) LctM-K159M for 1 h, or (D) LctM-K159M for 6 h.

Figure 5

MALDI-TOF mass spectra of assays of His₆-LctA with LctM-D242N and LctM-D259N. Substrates are shown in dashed line and assay product in solid line. (A, left) Dehydration assay with D242N, and (A, right) dehydration assay with LctM-D259N. (B) Phosphate elimination assay of a truncated, phosphorylated LctA analog reacted with LctM-D242N in the presence of ADP and Mg²⁺ resulting in a M-98 peak indicative of phosphate elimination. (C) Phosphate elimination assay of LctM-D259N.

Figure 6

MALDI-TOF mass spectra of assays of various peptides with LctM-R399M. Substrates are depicted in dashed line and assay products in solid line. R399M was incubated with His₆-LctA N39R/F45H (A), and His₆-LctA(1-37) (B) for 30 min. LctM-R399K was also incubated with His₆-LctA for 15 min (C). The peaks labeled with an asterisk (*) are showing single eliminations of phosphates from phosphorylated peptides.

Figure 7

MALDI-TOF mass spectra of phosphate elimination reactions. (A) His₆-LctA(1-37)S35A substrate is depicted in dotted line and phosphorylated substrate after 30 min incubation with R399M in dashed line. (B) Incubation of wild type LctM with phosphorylated LctA(1-37)S35A in the presence of 500 μM ADP and 10 mM Mg²⁺. (C) Incubation of wild type LctM with phosphorylated LctA(1-37)S35A in the presence of 500 μM ADP but in the absence of Mg²⁺.

Scheme 1

Proposed general mechanism of dehydration of Ser by LctM.