Secondary chemical shift analysis is the main NMR method for detection of transiently formed secondary structure in intrinsically disordered proteins. The quality of the secondary chemical shifts is dependent on an appropriate choice of random coil chemical shifts. We report random coil chemical shifts and sequence correction factors determined for a GGXGG peptide series following the approach of Schwarzinger et al. (J Am Chem Soc 123(13):2970-2978, 2001). The chemical shifts are determined at neutral pH in order to match the conditions of most studies of intrinsically disordered proteins. Temperature has a non-negligible effect on the (13)C random coil chemical shifts, so temperature coefficients are reported for the random coil chemical shifts to allow extrapolation to other temperatures. The pH dependence of the histidine random coil chemical shifts is investigated in a titration series, which allows the accurate random coil chemical shifts to be obtained at any pH. By correcting the random coil chemical shifts for the effects of temperature and pH, systematic biases of the secondary chemical shifts are minimized, which will improve the reliability of detection of transient secondary structure in disordered proteins.

Infection by the fungal pathogen Cryptococcus neoformans causes lethal meningitis, primarily in immune-compromised individuals. Colonization of the brain by C. neoformans is dependent on copper (Cu) acquisition from the host, which drives critical virulence mechanisms. While C. neoformans Cu + import and virulence are dependent on the Ctr1 and Ctr4 proteins, little is known concerning extracellular Cu ligands that participate in this process. We identified a C. neoformans gene, BIM1 , strongly induced during Cu limitation and which encodes a protein related to Lytic Polysaccharide Monooxygenases (LPMOs). Surprisingly, bim1 mutants are Cu deficient and Bim1 function in Cu accumulation depends upon Cu 2+ coordination and cell surface association via a GPI anchor. Bim1 participates in Cu uptake in concert with Ctr1 and expression of this pathway drives brain colonization in mouse infection models. These studies demonstrate a new role for LPMO-like proteins as a critical factor for Cu acquisition in fungal meningitis.

The limitations of fungal laccases at higher pH and salt concentrations have intensified the search for new extremophilic bacterial laccases. We report the cloning, expression, and characterization of the bacterial cotA from Bacillus clausii, a supposed alkalophilic ortholog of cotA from B. subtilis. Both laccases were expressed in E. coli strain BL21(DE3) and characterized fully in parallel for strict benchmarking. We report activity on ABTS, SGZ, DMP, caffeic acid, promazine, phenyl hydrazine, tannic acid, and bilirubin at variable pH. Whereas ABTS, promazine, and phenyl hydrazine activities vs. pH were similar, the activity of B. clausii cotA was shifted upwards by ∼0.5–2 pH units for the simple phenolic substrates DMP, SGZ, and caffeic acid. This shift is not due to substrate affinity (KM) but to pH dependence of catalytic turnover: The kcat of B. clausii cotA was 1 s−1 at pH 6 and 5 s−1 at pH 8 in contrast to 6 s−1 at pH 6 and 2 s−1 at pH 8 for of B. subtilis cotA. Overall, kcat/KM was 10-fold higher for B. subtilis cotA at pHopt. While both proteins were heat activated, activation increased with pH and was larger in cotA from B. clausii. NaCl inhibited activity at acidic pH, but not up to 500–700 mM NaCl in alkaline pH, a further advantage of the alkali regime in laccase applications. The B. clausii cotA had ∼20 minutes half-life at 80°C, less than the ∼50 minutes at 80°C for cotA from B. subtilis. While cotA from B. subtilis had optimal stability at pH∼8, the cotA from B. clausii displayed higher combined salt- and alkali-resistance. This resistance is possibly caused by two substitutions (S427Q and V110E) that could repel anions to reduce anion-copper interactions at the expense of catalytic proficiency, a trade-off of potential relevance to laccase optimization.

Lytic polysaccharide monooxygenase (LPMO) and copper binding protein CopC share a similar mononuclear copper site. This site is defined by an N-terminal histidine and a second internal histidine side chain in a configuration called the histidine brace. To understand better the determinants of reactivity, the biochemical and structural properties of a well-described cellulose-specific LPMO from Thermoascus aurantiacus (TaAA9A) is compared with that of CopC from Pseudomonas fluorescens (PfCopC) and with the LPMO-like protein Bim1 from Cryptococcus neoformans. PfCopC is not reduced by ascorbate but is a very strong Cu(II) chelator due to residues that interacts with the N-terminus. This first biochemical characterization of Bim1 shows that it is not redox active, but very sensitive to H2O2, which accelerates the release of Cu ions from the protein. TaAA9A oxidizes ascorbate at a rate similar to free copper but through a mechanism that produce fewer reactive oxygen species. These three biologically relevant examples emphasize the diversity in how the proteinaceous environment control reactivity of Cu with O2.

Lytic polysaccharide monooxygenases (LPMOs) are mononuclear copper enzymes that act in synergy with glycoside hydrolases to saccharify the most abundant polysaccharides in nature. Both O 2 and H 2 O 2 can be cosubstrates for LPMOs. The Lentinus similis LPMO (LsAA9A) has previously been shown to oxidatively cleave oligosaccharides when supplied with ascorbate as a reductant. This study demonstrates that LsAA9A is unable to complete the catalytic cycle and cleave cellulose without H 2 O 2 . Instead, cellooligomers efficiently prevent the slow continuous oxidation of ascorbate taking place under ambient conditions in the absence of a substrate. LsAA9A specifically cleaves cellooligomers in a fast and stoichiometric reaction with H 2 O 2 as a cosubstrate. However, the product profile contains more non-oxidized saccharides than anticipated by the generally accepted LPMO reaction scheme. The scission of glucosidic bonds and oxidation of the saccharide therefore appear not to be directly coupled. This was confirmed by the complete absence of oxidized products under anoxic conditions. A mechanism is proposed involving the hydrolysis of a cellulosic radical formed by a H 2 O 2 -derived caged hydroxyl radical. In addition, LsAA9A catalyzes another stoichiometric reaction with excess H 2 O 2 to oxidize ascorbate in the absence of cellulose. Ascorbate is not a cosubstrate for the reaction leading to the scission of glucosidic bonds by LsAA9A.