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Characterization of SLAC: A small laccase from Streptomyces coelicolor with unprecedented activity
Author(s) -
Machczynski Michael C.,
Vijgenboom Erik,
Samyn Bart,
Canters Gerard W.
Publication year - 2004
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.04759104
Subject(s) - laccase , streptomyces coelicolor , multicopper oxidase , chemistry , electron paramagnetic resonance , enzyme , copper , crystallography , stereochemistry , biochemistry , nuclear magnetic resonance , organic chemistry , physics , mutant , gene
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.