Distal Mutations Shape Substrate-Binding Sites during Evolution of a Metallo-Oxidase into a Laccase

Brissos V, Borges P, Núñez-Franco R, Lucas M, Frazão C, Monza E, Masgrau L, Cordeiro T, Martins L, ACS Catalysis :5022-5035 (2022) DOI

SASDHM8 – Aquifex aeolicus McoA metaloxidase ∆328-352 evolved variant (2F4∆328-352)

Aquifex aeolicus McoA metaloxidase ∆328-352 evolved variant (2F4∆328-352)
MWexperimental 53 kDa
MWexpected 53 kDa
VPorod 74 nm3
log I(s) 3.66×101 3.66×100 3.66×10-1 3.66×10-2
Aquifex aeolicus McoA metaloxidase ∆328-352 evolved variant (2F4∆328-352) small angle scattering data  s, nm-1
ln I(s)
Aquifex aeolicus McoA metaloxidase ∆328-352 evolved variant (2F4∆328-352) Guinier plot ln 3.66×101 Rg: 2.2 nm 0 (2.2 nm)-2 s2
(sRg)2I(s)/I(0)
Aquifex aeolicus McoA metaloxidase ∆328-352 evolved variant (2F4∆328-352) Kratky plot 1.104 0 √3 sRg
p(r)
Aquifex aeolicus McoA metaloxidase ∆328-352 evolved variant (2F4∆328-352) pair distance distribution function Rg: 2.2 nm 0 Dmax: 6.6 nm

Data validation

Fits and models

log I(s)
 s, nm-1
Aquifex aeolicus McoA metaloxidase ∆328-352 evolved variant (2F4∆328-352) DAMMIF model
log I(s)
 s, nm-1
Aquifex aeolicus McoA metaloxidase ∆328-352 evolved variant (2F4∆328-352) ROSETTA model
Synchrotron SAXS data from solutions of Aquifex aeolicus McoA metaloxidase ∆328-352 evolved variant (2F4∆328-352) in 50 mM Tris-HCl, 150 mM NaCl, 2 mM TCEP, pH 7.5 were collected on the BM29 beam line at the ESRF (Grenoble, France) using a Pilatus 1M detector at a sample-detector distance of 2.9 m and at a wavelength of λ = 0.099 nm (I(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). In-line size-exclusion chromatography (SEC) SAS was employed. The SEC parameters were as follows: A 250 μl sample at 10 mg/ml was injected at a 0.50 ml/min flow rate onto a GE Superdex 200 Increase 10/300 column at 20°C. 3000 successive 1 second frames were collected through the SEC elution. The data were normalized to the intensity of the transmitted beam and radially averaged; the scattering of the solvent-blank was subtracted from the sample peak data frames.

The scattering intensity from the monomeric elution single-peak region were integrated and buffer subtracted to produce the SAXS-profile of 2F4 using the ScÅtter software. Further processing was performed using the ATSAS software suite.

Aquifex aeolicus McoA metaloxidase ∆328-352 evolved variant (2F4∆328-352) (2F4∆328-352)
Mol. type   Protein
Organism   Aquifex aeolicus
Olig. state   Monomer
Mon. MW   52.7 kDa
Sequence   FASTA
 
PDB ID   6TTD