Crystal structure of the flavin reductase of Acinetobacter baumannii p-hydroxyphenylacetate 3-hydroxylase (HPAH) and identification of amino acid residues underlying its regulation by aromatic ligands.

Yuenyao A, Petchyam N, Kamonsutthipaijit N, Chaiyen P, Pakotiprapha D
Arch Biochem Biophys 653:24-38 (2018 Sep 1)

PMID: 29940152 doi: 10.1016/j.abb.2018.06.010

Submitted to SASBDB: 2018 Jun 5 Published in SASBDB: 2018 Jun 29

SASDDN7 – p-hydroxyphenylacetate 3-hydroxylase, reductase component (mutant): 2 mg/ml of E248A/E251A C1 in the absence of p-hydroxyphenylacetic acid (HPA) Sample: P-hydroxyphenylacetate 3-hydroxylase (HPAH), reductase component E248A/E251A C1 dimer, 71 kDa Acinetobacter baumannii protein Buffer: 50 mM MOPS, 0.5 mM EDTA, 1 mM DTT, 50 mM NaCl, and 5% glycerol, pH: 7 Experiment: SAXS data collected at BL1.3W, Synchrotron Light Research Institute (SLRI) on 2018 May 26 RgGuinier 2.3 nm Dmax 6.7 nm VolumePorod 88 nm3

SASDDP7 – p-hydroxyphenylacetate 3-hydroxylase, reductase component (mutant): 4 mg/ml of E248A/E251A C1 in the absence of p-hydroxyphenylacetic acid (HPA) Sample: P-hydroxyphenylacetate 3-hydroxylase (HPAH), reductase component E248A/E251A C1 dimer, 71 kDa Acinetobacter baumannii protein Buffer: 50 mM MOPS, 0.5 mM EDTA, 1 mM DTT, 50 mM NaCl, and 5% glycerol, pH: 7 Experiment: SAXS data collected at BL1.3W, Synchrotron Light Research Institute (SLRI) on 2018 May 26 RgGuinier 2.4 nm Dmax 6.8 nm VolumePorod 90 nm3

SASDDQ7 – p-hydroxyphenylacetate 3-hydroxylase, reductase component (mutant): 8 mg/ml of E248A/E251A C1 in the absence of p-hydroxyphenylacetic acid (HPA) Sample: P-hydroxyphenylacetate 3-hydroxylase (HPAH), reductase component E248A/E251A C1 dimer, 71 kDa Acinetobacter baumannii protein Buffer: 50 mM MOPS, 0.5 mM EDTA, 1 mM DTT, 50 mM NaCl, and 5% glycerol, pH: 7 Experiment: SAXS data collected at BL1.3W, Synchrotron Light Research Institute (SLRI) on 2018 May 26 RgGuinier 2.4 nm Dmax 7.1 nm VolumePorod 95 nm3

SASDDR7 – p-hydroxyphenylacetate 3-hydroxylase, reductase component (mutant): 2 mg/ml of E248A/E251A C1 in the presence of 1 mM p-hydroxyphenylacetic acid (HPA) Sample: P-hydroxyphenylacetate 3-hydroxylase (HPAH), reductase component E248A/E251A C1 dimer, 71 kDa Acinetobacter baumannii protein Buffer: 50 mM MOPS, 0.5 mM EDTA, 1 mM DTT, 50 mM NaCl, 1 mM HPA, and 5% glycerol, pH: 7 Experiment: SAXS data collected at BL1.3W, Synchrotron Light Research Institute (SLRI) on 2018 May 26 RgGuinier 2.5 nm Dmax 8.1 nm VolumePorod 83 nm3

SASDDS7 – p-hydroxyphenylacetate 3-hydroxylase, reductase component (mutant): 4 mg/ml of E248A/E251A C1 in the presence of 1 mM p-hydroxyphenylacetic acid (HPA) Sample: P-hydroxyphenylacetate 3-hydroxylase (HPAH), reductase component E248A/E251A C1 dimer, 71 kDa Acinetobacter baumannii protein Buffer: 50 mM MOPS, 0.5 mM EDTA, 1 mM DTT, 50 mM NaCl, 1 mM HPA, and 5% glycerol, pH: 7 Experiment: SAXS data collected at BL1.3W, Synchrotron Light Research Institute (SLRI) on 2018 May 26 RgGuinier 2.6 nm Dmax 8.5 nm VolumePorod 83 nm3

SASDDT7 – p-hydroxyphenylacetate 3-hydroxylase, reductase component (mutant): 8 mg/ml of E248A/E251A C1 in the presence of 1 mM p-hydroxyphenylacetic acid (HPA) Sample: P-hydroxyphenylacetate 3-hydroxylase (HPAH), reductase component E248A/E251A C1 dimer, 71 kDa Acinetobacter baumannii protein Buffer: 50 mM MOPS, 0.5 mM EDTA, 1 mM DTT, 50 mM NaCl, 1 mM HPA, and 5% glycerol, pH: 7 Experiment: SAXS data collected at BL1.3W, Synchrotron Light Research Institute (SLRI) on 2018 May 26 RgGuinier 2.7 nm Dmax 8.8 nm VolumePorod 88 nm3

SASDDU7 – p-hydroxyphenylacetate 3-hydroxylase, reductase component (mutant): 2 mg/ml of E248A C1 in the absence of p-hydroxyphenylacetic acid (HPA) Sample: P-hydroxyphenylacetate 3-hydroxylase (HPAH), reductase component E248A mutant dimer, 71 kDa Acinetobacter baumannii protein Buffer: 50 mM MOPS, 0.5 mM EDTA, 1 mM DTT, 50 mM NaCl, 10 % glycerol, pH: 7 Experiment: SAXS data collected at BL1.3W, Synchrotron Light Research Institute (SLRI) on 2018 Apr 25 RgGuinier 2.5 nm Dmax 7.9 nm VolumePorod 96 nm3

SASDDV7 – p-hydroxyphenylacetate 3-hydroxylase, reductase component (mutant): 4 mg/ml of E248A C1 in the absence of p-hydroxyphenylacetic acid (HPA) Sample: P-hydroxyphenylacetate 3-hydroxylase (HPAH), reductase component E248A mutant dimer, 71 kDa Acinetobacter baumannii protein Buffer: 50 mM MOPS, 0.5 mM EDTA, 1 mM DTT, 50 mM NaCl, 10 % glycerol, pH: 7 Experiment: SAXS data collected at BL1.3W, Synchrotron Light Research Institute (SLRI) on 2018 Apr 25 RgGuinier 2.6 nm Dmax 8.1 nm VolumePorod 97 nm3

SASDDW7 – p-hydroxyphenylacetate 3-hydroxylase, reductase component (mutant): 8 mg/ml of E248A C1 in the absence of p-hydroxyphenylacetic acid (HPA) Sample: P-hydroxyphenylacetate 3-hydroxylase (HPAH), reductase component E248A mutant dimer, 71 kDa Acinetobacter baumannii protein Buffer: 50 mM MOPS, 0.5 mM EDTA, 1 mM DTT, 50 mM NaCl, 10 % glycerol, pH: 7 Experiment: SAXS data collected at BL1.3W, Synchrotron Light Research Institute (SLRI) on 2018 Apr 25 RgGuinier 2.5 nm Dmax 7.9 nm VolumePorod 94 nm3

SASDDX7 – p-hydroxyphenylacetate 3-hydroxylase, reductase component (mutant): 2 mg/ml of E248A C1 in 1 mM of p-hydroxyphenylacetic acid (HPA) Sample: P-hydroxyphenylacetate 3-hydroxylase (HPAH), reductase component E248A mutant dimer, 71 kDa Acinetobacter baumannii protein Buffer: 50 mM MOPS, 0.5 mM EDTA, 1 mM DTT, 50 mM NaCl, 1 mM HPA, 10 % glycerol, pH: 7 Experiment: SAXS data collected at BL1.3W, Synchrotron Light Research Institute (SLRI) on 2018 Apr 25 RgGuinier 2.7 nm Dmax 9.0 nm VolumePorod 94 nm3

SASDDY7 – p-hydroxyphenylacetate 3-hydroxylase, reductase component (mutant): 4 mg/ml of E248A C1 in 1 mM of p-hydroxyphenylacetic acid (HPA) Sample: P-hydroxyphenylacetate 3-hydroxylase (HPAH), reductase component E248A mutant dimer, 71 kDa Acinetobacter baumannii protein Buffer: 50 mM MOPS, 0.5 mM EDTA, 1 mM DTT, 50 mM NaCl, 1 mM HPA, 10 % glycerol, pH: 7 Experiment: SAXS data collected at BL1.3W, Synchrotron Light Research Institute (SLRI) on 2018 Apr 25 RgGuinier 2.8 nm Dmax 9.0 nm VolumePorod 91 nm3

SASDDZ7 – p-hydroxyphenylacetate 3-hydroxylase, reductase component (mutant): 8 mg/ml of E248A C1 in 1 mM of p-hydroxyphenylacetic acid (HPA) Sample: P-hydroxyphenylacetate 3-hydroxylase (HPAH), reductase component E248A mutant dimer, 71 kDa Acinetobacter baumannii protein Buffer: 50 mM MOPS, 0.5 mM EDTA, 1 mM DTT, 50 mM NaCl, 1 mM HPA, 10 % glycerol, pH: 7 Experiment: SAXS data collected at BL1.3W, Synchrotron Light Research Institute (SLRI) on 2018 Apr 25 RgGuinier 2.7 nm Dmax 8.8 nm VolumePorod 91 nm3

SASDD28 – p-hydroxyphenylacetate 3-hydroxylase, reductase component (mutant): 2 mg/ml of E251A C1 in the absence of p-hydroxyphenylacetic acid (HPA) Sample: P-hydroxyphenylacetate 3-hydroxylase (HPAH), reductase component E251A mutant dimer, 71 kDa Acinetobacter baumannii protein Buffer: 50 mM MOPS, 0.5 mM EDTA, 1 mM DTT, 50 mM NaCl, 10 % glycerol, pH: 7 Experiment: SAXS data collected at BL1.3W, Synchrotron Light Research Institute (SLRI) on 2018 Apr 25 RgGuinier 2.6 nm Dmax 8.4 nm VolumePorod 89 nm3

SASDD38 – p-hydroxyphenylacetate 3-hydroxylase, reductase component (mutant): 4 mg/ml of E251A C1 in the absence of p-hydroxyphenylacetic acid (HPA) Sample: P-hydroxyphenylacetate 3-hydroxylase (HPAH), reductase component E251A mutant dimer, 71 kDa Acinetobacter baumannii protein Buffer: 50 mM MOPS, 0.5 mM EDTA, 1 mM DTT, 50 mM NaCl, 10 % glycerol, pH: 7 Experiment: SAXS data collected at BL1.3W, Synchrotron Light Research Institute (SLRI) on 2018 Apr 25 RgGuinier 2.7 nm Dmax 8.9 nm VolumePorod 93 nm3

SASDD48 – p-hydroxyphenylacetate 3-hydroxylase, reductase component (mutant): 8 mg/ml of E251A C1 in the absence of p-hydroxyphenylacetic acid (HPA) Sample: P-hydroxyphenylacetate 3-hydroxylase (HPAH), reductase component E251A mutant dimer, 71 kDa Acinetobacter baumannii protein Buffer: 50 mM MOPS, 0.5 mM EDTA, 1 mM DTT, 50 mM NaCl, 10 % glycerol, pH: 7 Experiment: SAXS data collected at BL1.3W, Synchrotron Light Research Institute (SLRI) on 2018 Apr 25 RgGuinier 2.7 nm Dmax 8.8 nm VolumePorod 92 nm3

SASDD58 – p-hydroxyphenylacetate 3-hydroxylase, reductase component (mutant): 2 mg/ml of E251A C1 in 1 mM of p-hydroxyphenylacetic acid (HPA) Sample: P-hydroxyphenylacetate 3-hydroxylase (HPAH), reductase component E251A mutant dimer, 71 kDa Acinetobacter baumannii protein Buffer: 50 mM MOPS, 0.5 mM EDTA, 1 mM DTT, 50 mM NaCl, 1 mM HPA, 10 % glycerol, pH: 7 Experiment: SAXS data collected at BL1.3W, Synchrotron Light Research Institute (SLRI) on 2018 Apr 25 RgGuinier 2.8 nm Dmax 8.9 nm VolumePorod 86 nm3

SASDD68 – p-hydroxyphenylacetate 3-hydroxylase, reductase component (mutant): 4 mg/ml of E251A C1 in 1 mM of p-hydroxyphenylacetic acid (HPA) Sample: P-hydroxyphenylacetate 3-hydroxylase (HPAH), reductase component E251A mutant dimer, 71 kDa Acinetobacter baumannii protein Buffer: 50 mM MOPS, 0.5 mM EDTA, 1 mM DTT, 50 mM NaCl, 1 mM HPA, 10 % glycerol, pH: 7 Experiment: SAXS data collected at BL1.3W, Synchrotron Light Research Institute (SLRI) on 2018 Apr 25 RgGuinier 2.9 nm Dmax 9.5 nm VolumePorod 82 nm3

SASDD78 – p-hydroxyphenylacetate 3-hydroxylase, reductase component (mutant): 8 mg/ml of E251A C1 in 1 mM of p-hydroxyphenylacetic acid (HPA) Sample: P-hydroxyphenylacetate 3-hydroxylase (HPAH), reductase component E251A mutant dimer, 71 kDa Acinetobacter baumannii protein Buffer: 50 mM MOPS, 0.5 mM EDTA, 1 mM DTT, 50 mM NaCl, 1 mM HPA, 10 % glycerol, pH: 7 Experiment: SAXS data collected at BL1.3W, Synchrotron Light Research Institute (SLRI) on 2018 Apr 25 RgGuinier 2.9 nm Dmax 9.3 nm VolumePorod 89 nm3