Involvement of Tyr108 in the Enzyme Mechanism of the Small Laccase from Streptomyces coelicolor

Abstract

The enzyme mechanism of the multicopper oxidase (MCO) SLAC from Streptomyces coelicolor was investigated by structural (XRD), spectroscopic (optical, EPR), and kinetics (stopped-flow) experiments on variants in which residue Tyr108 had been replaced by Phe or Ala through site-directed mutagenesis. Contrary to the more common three-domain MCOs, a tyrosine in the two-domain SLAC is found to participate in the enzyme mechanism by providing an electron during oxygen reduction, giving rise to the temporary appearance of a tyrosyl radical. The relatively low k(cat)/K(M) of SLAC and the involvement of Y108 in the enzyme mechanism may reflect an adaptation to a milieu in which there is an imbalance between the available reducing and oxidizing co-substrates. The purported evolutionary relationship between the two-domain MCOs and human ceruloplasmin appears to extend not only to the 3D structure and the mode of binding of the Cu's in the trinuclear center, as noted before, but also to the enzyme mechanism.

Biochemical studies of the multicopper oxidase (small laccase) from Streptomyces coelicolor using bioactive phytochemicals and site‐directed mutagenesis

“…The published crystal structure of SLAC revealed that the two domain protein forms trimers in solution, where each trimer contains three trinuclear copper clusters (types 2 and 3) and three mononuclear copper centres (type‐1) (Skálová et al ., ). Site‐directed mutagenesis was recently used to investigate the role of Tyr108 in the reduction of oxygen, and revealed the potential of this tyrosine residue to minimize the lifetime of reactive oxygen species by providing an additional source of reductant (Gupta et al ., ). In the current study, 17 amino acids were individually mutated to Ala to identify the residues important for SLAC activity, as well as to confirm key residues involved in copper binding, and to predict those that could be targeted to alter the substrate specificity of this enzyme (Table S1, Fig.…”

A model of the transient kinetics of laccase-catalyzed oxidation of phenol at micromolar concentrations

“…It was also reported that the formation of E RO is reversible to E* [66], as is indicated in Figure 1. Both E* and E RO react with substrates in four sequential single electron reduction steps to form reduced enzyme, E, and substrate radicals along with a single molecule of H 2 O from E RO and two from E* [65,85,86]. The formation of E RO and its subsequent reduction to E are considered too slow to play a significant part in catalytic cycle [63][64][65].…”

“…The electron transfer between the two sites is in the direction from T1 to T3. The reduction of O 2 and intermediate forms of laccase within the catalytic cycle was studied in detail by several research groups[63][64][65][66][85][86][87] who identified at least four intermediates. The binding of O 2 to fully reduced laccase, E, with the resulting two electron reduction of O 2 results in the formation of a peroxide intermediate, E P , which is then followed by a second two electron reduction step to form the oxidized enzyme, E*.…”

“…The binding of O 2 to fully reduced laccase, E, with the resulting two electron reduction of O 2 results in the formation of a peroxide intermediate, E P , which is then followed by a second two electron reduction step to form the oxidized enzyme, E*. The rate limiting step in the reaction is the formation of the E*[65,85,86]. Once fully oxidized to E*, a slow decay can occur involving the addition of two protons (H + ), by internal rearrangement among the trinuclear cluster.…”

The effect of mutations near the T1 copper site on the biochemical characteristics of the small laccase from Streptomyces coelicolor A3(2)

“…It is, however, interesting to note that the "typical" laccase inhibitor, NaN 3, which disrupts the flow of electrons at the T2 and T3 coppers, had no inhibitory effect up to 5 mM on SLAC-M298F, and no significant inhibitory effect on the other variants at a concentration of 0.1 mM [4]. It is suggested that slight differences in the copper coordination of the trinuclear clusters of SLAC, compared to 25 typical three-domain laccases, may be responsible for a difference in the flow of electrons when molecular oxygen is reduced, which may be why SLAC and the mutant variants are resistant to complete inhibition by NaN 3[13,41].…”