Investigations of Accessibility of T2/T3 Copper Center of Two-Domain Laccase from Streptomyces griseoflavus Ac-993

Abstract

Laccases (EC 1.10.3.2) are multicopper oxidoreductases acting on diphenols and related substances. Laccases are highly important for biotechnology and environmental remediation. These enzymes contain mononuclear one T2 copper ion and two T3 copper ions (Cu3α and Cu3β), which form the so-called trinuclear center (TNC). Along with the typical three-domain laccases Bacteria produce two-domain (2D) enzymes, which are active at neutral and basic pH, thermostable, and resistant to inhibitors. In this work we present the comparative analysis of crystal structures and catalytic properties of recombinant 2D laccase from Streptomyces griseoflavus Ac-993 (SgfSL) and its four mutant forms with replacements of two amino acid residues, located at the narrowing of the presumable T3-solvent tunnels. We obtained inactive enzymes with substitutions of His165, with Phe, and Ile170 with Ala or Phe. His165Ala variant was more active than the wild type. We suggest that His165 is a “gateway” at the O2-tunnel leading from solvent to the Cu3β of the enzyme. The side chain of Ile170 could be indirectly involved in the coordination of copper ions at the T3 center by maintaining the position of the imidazole ring of His157 that belongs to the first coordination sphere of Cu3α.

The Resting Oxidized State of Small Laccase Analyzed with Paramagnetic NMR Spectroscopy

“…This water channel has been proposed to be the source of protons in the formation of waters from dioxygen. [21,25] Gln291 also forms a strong hydrogen bond with the Nδ1 of His289 (Figure 2c), a ligand in the T3 site.…”

Applications of Microbial Laccases: Patent Review of the Past Decade (2009–2019)

“…PDB entries corresponding to the term 'laccase' have been reviewed previously by [9]. [32] The molecular architecture of most laccases includes three cupredoxin domains with a β-barrel structure, although laccases with two such domains have been discovered, which are named two-domain or Small Laccases (SLACs, [20]). In a typical structure, a mononuclear copper-binding site T1 is situated in domain 3 close to the protein surface, and a trinuclear copper-binding site (TNC) consisting of one T2 and two T3 copper atoms is formed by the proximity of domains 1 and 3 (Figure 1).…”

Supplementary material to "Towards resolving the complex paramagnetic NMR spectrum of small laccase: Assignments of resonances to residue specific nuclei"

“…Two H-bond acceptors for His236. (a) Overlayed TNC from crystal structure 3cg8 in gold and 6s0o in blue highlighting the hydrogen bond of the Nd1 from the T3 histidine ligand His236/237 (Gabdulkhakov et al, 2019;Skálová et al, 2009). The c2 dihedral angle for histidine 236/237 is shown for both crystal structures.…”

“…The c2 dihedral angle for histidine 236/237 is shown for both crystal structures. Hydrogen bonds are shown as red lines; (b) Protons are modelled for the crystal structure 6s0o using the algorithm as implemented in UCSF Chimera (Gabdulkhakov et al, 2019;Pettersen et al, 2004). The values of the angles between [Asp114 Od1 -His237 Nd1 - (Dasgupta et al, 2020) while the 1 H resonance of 17 and 18 are broad (~ 1.1 ppm), which can affect the evolution period d2 in the 1 H-15 N HMQC ( Figure S2).…”