Characterization of SLAC: a small laccase from Streptomyces coelicolor with unprecedented activity.
Laccases and other four-copper oxidases are usually constructed of three domains: Domains one and three house the copper sites, and the second domain often helps form a substrate-binding cleft. In contrast to this arrangement, the genome of Streptomyces coelicolor was found to encode a small, four-copper oxidase that lacks the second domain. This protein is representative of a new family of enzymes--the two-domain laccases. Disruption of the corresponding gene abrogates laccase activity in the growth media. We have recombinantly expressed this enzyme, called SLAC, in Escherichia coli and characterized it. The enzyme binds four copper ions/monomer, and UV-visible absorption and EPR measurements confirm that the conserved type 1 copper site and trinuclear cluster are intact. We also report the first known paramagnetic NMR spectrum for the trinuclear copper cluster of a protein from the laccase family. The enzyme is highly stable, retaining activity as a dimer in denaturing gels after boiling and SDS treatment. The activity of the enzyme against 2,6-dimethoxyphenol (DMP) peaks at an unprecedentedly high pH (9.4), whereas the activity against ferrocyanide decreases with pH. SLAC binds negatively charged substrates more tightly than positively charged or uncharged molecules.
Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, 2333 CC, Leiden, The Netherlands.
Michael C
MC
Machczynski
Erik
E
Vijgenboom
Bart
B
Samyn
Gerard W
GW
Canters
D000595:Amino Acid Sequence; D001483:Base Sequence; D003300:Copper / Q000378:metabolism; D003902:Detergents / Q000737:chemistry; D019281:Dimerization; D004578:Electron Spin Resonance Spectroscopy; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005782:Gels; D006863:Hydrogen-Ion Concentration; D042845:Laccase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D011748:Pyrogallol / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet; D048372:Streptomyces coelicolor / Q000201:enzymology* / Q000235:genetics
D003902:Detergents; D005782:Gels; D011994:Recombinant Proteins; D011748:Pyrogallol; D012967:Sodium Dodecyl Sulfate; C010120:pyrogallol 1,3-dimethyl ether; D003300:Copper; D042845:Laccase