How pH Modulates the Reactivity and Selectivity of a Siderophore-Associated Flavin Monooxygenase

Flavin-containing monooxygenases (FMOs) catalyze the oxygenation of diverse organic molecules using O₂, NADPH, and the flavin adenine dinucleotide (FAD) cofactor. The fungal FMO SidA initiates peptidic siderophore biosynthesis via the highly selective hydroxylation of l-ornithine, while the related amino acid l-lysine is a potent effector of reaction uncoupling to generate H₂O₂. We hypothesized that protonation states could critically influence both substrate-selective hydroxylation and H₂O₂ release, and therefore undertook a study of SidA’s pH-dependent reaction kinetics. Consistent with other FMOs that stabilize a C4a-OO­(H) intermediate, SidA’s reductive half reaction is pH independent. The rate constant for the formation of the reactive C4a-OO­(H) intermediate from reduced SidA and O₂ is likewise independent of pH. However, the rate constants for C4a-OO­(H) reactions, either to eliminate H₂O₂ or to hydroxylate l-Orn, were strongly pH-dependent and influenced by the nature of the bound amino acid. Solvent kinetic isotope effects of 6.6 ± 0.3 and 1.9 ± 0.2 were measured for the C4a-OOH/H₂O₂ conversion in the presence and absence of l-Lys, respectively. A model is proposed in which l-Lys accelerates H₂O₂ release via an acid–base mechanism and where side-chain position determines whether H₂O₂ or the hydroxylation product is observed

Identification of MPUS re-annotates approximately 430 proteins.

<p>Each node represents one of the protein sequences identified as homologous to MPUS from <i>Micrococcus luteus</i>; edges between nodes are drawn only if the similarity between a pair of sequences is better than an E-value threshold cutoff of 1e⁻⁵⁰. The network is visualized using the organic layout in Cytoscape. Nodes are colored according to assigned function in UniProtKB database; megenta: DPM1, blue: apolipoprotein N-acyltransferase Lnt, red: Ppm1, cyan: MPUS. Larger nodes are proteins that are used to generate sequence alignment. Large rectangular nodes with green borders are proteins with evidence of existence at the protein level.</p

Structural superimposition between a model structure of maltose epimerase and structures of its homologous enzymes shows conservation of active site residues.

<p>Red: Galactose 1-epimerase from <i>Lactobacillus acidophilus</i> pdb:3imh; magenta: human galactose mutarotase pdb:1snz; yellow: galactose mutarotase/UDP-galactose 4-epimerase from <i>Saccharomyces cerevisiae</i> pdb:1z45; blue: galactose mutarotase from <i>Lactococcus lactis</i> pdb:1l7j; cayan: model structure of maltose epimerase. The numbering is according to galactose mutarotase from <i>Lactococcus lactis</i>.</p

Identification of FDH-I re-annotates 160 proteins.

<p>Proteins with sequences similar to the newly identified FDH-I were clustered based on their internal sequence similarities. Each node represents one of the protein sequences identified as homologous to fructose dehydrogenase from <i>Gluconobacter frateurii</i>; edges between nodes are drawn only if the similarity between a pair of sequences is better than an E-value threshold cutoff of 1e⁻⁶⁶. The network is visualized using the organic layout in Cytoscape. Nodes are colored according to assigned function in UniProtKB database; yellow: 2-keto-gluconate dehydrogenase, blue: choline dehydrogenase, cyan: fructose dehydrogenase from <i>Gluconobacter frateurii,</i> red: gluconate dehydrogenase, green: glucose-methanol-choline dehydrogenase. Large diamond shaped nodes with red borders are proteins that are used for identifying gene proximity clusters. Larger nodes are proteins with evidence of existence at the protein level.</p

FDH-I homologs retain their genome context.

<p>The species of origin of the gene clusters are indicated at the top of each. Genes with orthologs in other organisms are colored similarly (except grey which are genes without any detectable homolog in other genomes). Genes for fructose dehydrogenase, cytochrome c and ∼19 kDa uncharacterized protein are indicated at the bottom.</p

“Filtering” by MW allows rapid MS-based proteomic identification of mannosylphosphorylundecaprenol synthase.

<p>(A) 1-D SDS PAGE of a partially purified <i>M. luteus</i> protein fraction exhibiting MPU synthase activity reveals multiple protein bands ranging from 26–35 kDa. The indicated band (boxed) was excised for MS analysis. (B) MS/MS fragmentation analysis of a doubly charged peptide observed at 598.81 m/z identifies the tryptic peptide DGLGGAYIAGFR from a glycosyl transferase protein in <i>M. luteus</i>. (C) Complete amino acid sequence of a DPM1-like glycosyl transferase identified in queries against a custom <i>M. luteus</i> protein database. High confidence (>90%) MS-identified peptides are colored green. Additional peptide matches are colored red.</p

“Filtering” by MW allows rapid MS-based proteomic identification of maltose epimerase.

<p>(A) 1-D SDS PAGE of maltose epimerase (ME) (Sigma Aldrich #M0902) resolves as a band at ∼40 kDa (boxed), the expected molecular weight for maltose epimerase. (B) Tryptic digest and MS analysis of the ∼40 kDa protein band yields a prominent doubly charged precursor ion peak at 727.84 m/z. (C) MS/MS fragmentation analysis of the 727.84 m/z peptide identified as the tryptic peptide DTPIATIGDTTGHR from the <i>L. brevis</i> protein aldose 1-epimerase. b-ion (green) and y-ion (red) series are shown. (D) Complete amino acid sequence of <i>L. brevis</i> protein aldose 1-epimerase with high confidence (>95%) MS-identified peptides colored green. Additional peptide matches are colored red.</p

Mapping onto a related organism allows rapid MS-based proteomic identification of fructose 5-dehydrogenase from the unsequenced organism <i>Gluconobacter industrius.</i>

<p>MS-based proteomic identification of fructose 5-dehydrogenase. (A) 1-D SDS PAGE of 140 ug of fructose 5-dehydrogenase (F5D) (Sigma Aldrich: #F4892) yields 7 distinct protein bands after Coomassie staining. A band at ∼75 kDa (boxed) was excised for MS analysis. (B) Zoomed-in mass spectra illustrating the triply charged precursor ion at 710.67 m/z observed in MS scans subsequently identified through MS/MS as the tryptic peptide DALGIPHPEVTYDVGEYVR from fructose dehydrogenase large subunit protein in <i>G. frateurri</i>. (C) Complete amino acid sequence of the <i>G. frateurri</i> fructose dehydrogenase large subunit protein identified in protein database searches. High confidence (>90%) MS-identified peptides are colored green. Additional peptide matches are colored red.</p

Active site residues are well conserved across MPUS homologs.

<p>Sequence alignment was generated using MAFFT alignment program. Residues that are conserved in more than 95% species are highlighted in black, residues that are conserved in more than 80% species are highlighted in dark grey and residues that are conserved in more than 50% species are highlighted in grey. Residues that have been experimentally shown to be functionally important are denoted with asterisks on the top and residues proposed to be important for the function are denoted with asterisks at the bottom.</p