Structural and Mechanistic Insights into Caffeine Degradation by the Bacterial N-Demethylase Complex.

Kim JH, Kim BH, Brooks S, Kang SY, Summers RM, Song HK, J Mol Biol 431(19):3647-3661 (2019) Europe PMC

SASDFH7 – Pseudomonas putida CBB5 NdmAB complex

Methylxanthine N1-demethylase NdmA
Methylxanthine N3-demethylase NdmB
MWexperimental 227 kDa
MWexpected 256 kDa
log I(s) 2.72×100 2.72×10-1 2.72×10-2 2.72×10-3
Methylxanthine N1-demethylase NdmA Methylxanthine N3-demethylase NdmB small angle scattering data  s, nm-1
ln I(s)
Methylxanthine N1-demethylase NdmA Methylxanthine N3-demethylase NdmB Guinier plot ln 2.72×100 Rg: 4.2 nm 0 (4.2 nm)-2 s2
(sRg)2I(s)/I(0)
Methylxanthine N1-demethylase NdmA Methylxanthine N3-demethylase NdmB Kratky plot 1.104 0 √3 sRg
p(r)
Methylxanthine N1-demethylase NdmA Methylxanthine N3-demethylase NdmB pair distance distribution function Rg: 4.1 nm 0 Dmax: 10.9 nm

Data validation

Fits and models

log I(s)
 s, nm-1
Methylxanthine N1-demethylase NdmA Methylxanthine N3-demethylase NdmB DAMMIF model
Synchrotron SAXS data from solutions of Pseudomonas putida CBB5 NdmAB complex in 20 mM HEPES 150 mM NaCl 2 mM TCEP, pH 7.5 were collected on the BL-10C beam line at the Photon Factory (PF), High Energy Accelerator Research Organization (KEK) storage ring (Tsukuba, Japan) using a Pilatus3 2M detector at a sample-detector distance of 3 m and at a wavelength of λ = 0.15 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 200.00 μl sample at 21.6 mg/ml was injected at a 0.05 ml/min flow rate onto a GE Superdex 200 Increase 10/300 column at 18°C. 48 successive 10 second frames were collected. The data were normalized to the intensity of the transmitted beam and radially averaged; the scattering of the solvent-blank was subtracted.

Methylxanthine N1-demethylase NdmA
Mol. type   Protein
Organism   Pseudomonas putida
Olig. state   Trimer
Mon. MW   42.3 kDa
 
UniProt   H9N289
Sequence   FASTA
 
Methylxanthine N3-demethylase NdmB
Mol. type   Protein
Organism   Pseudomonas putida
Olig. state   Trimer
Mon. MW   43.0 kDa
 
UniProt   H9N290
Sequence   FASTA