AdaV

Enzyme Information
reference: 10.1002/anie.201914994
host: E. coli BL21 (DE3)
note: chlorinates at the C2′ position of 2′-deoxyadenosine monophosphate and accepts 2′,3′-dideoxyadenosine-5′-monophosphate and 2′-deoxyinosine-5′-monophosphate as well
test: HPLC analysis with knockout; incubation of AdeV with substrates, iron and alpha-ketoglutarate and tandem MS
producer: Actinomadura sp. ATCC 39365
phmm-model: NHFe_nucleotide.hmm

Gene Data
phmm effect mutation gene_name reference
63 phosphate binding pocket R237 AdaV DOI: 10.1021/acscatal.2c04608 DOI: 10.1038/s41467-024-49147-7
- close to substrate binding site K107 AdaV DOI: 10.1038/s41467-024-49147-7
128 phosphate binding site R302 AdaV DOI: 10.1038/s41467-024-49147-7
22 creates iron coordination site for chloride binding; completely conserved in homologs G196 AdaV DOI: 10.1021/acscatal.2c04608
- lost halogeantion activity; reduced hydroxylation activity Q203A&V269A&G196E AdaV DOI: 10.1021/acscatal.2c04608
- abolished ahlogenation activity H194A AdaV DOI: 10.1021/acscatal.2c04608
- abolished ahlogenation activity H252A AdaV DOI: 10.1021/acscatal.2c04608
- lost halogeantion activity; reduced hydroxylation activity Q203A&V269A&G196A AdaV DOI: 10.1021/acscatal.2c04608
60 None N234 AdaV DOI: 10.1021/acscatal.2c04608
27 None G201 AdaV DOI: 10.1021/acscatal.2c04608
- close to substrate binding site D105 AdaV DOI: 10.1038/s41467-024-49147-7
23 substrate binding pocket I197 AdaV DOI: 10.1021/acscatal.2c04608
- substrate binding pocket; H-bond network P106 AdaV DOI: 10.1021/acscatal.2c04608
8 substrate binding pocket; H-bond network R182 AdaV DOI: 10.1021/acscatal.2c04608
24 substrate binding pocket; H-bond with deoxyribose moiety Q198 AdaV DOI: 10.1021/acscatal.2c04608
- abolished halogenation activity I197A AdaV DOI: 10.1021/acscatal.2c04608
29 orient Fe(III)-OH intermediate to constrain the O rebounding onto the radical substrate; important to supress hydroxylation; 2OG orientation Q203 AdaV DOI: 10.1021/acscatal.2c04608
95 orient Fe(III)-OH intermediate to constrain the O rebounding onto the radical substrate; important to supress hydroxylation; 2OG orientation V269 AdaV DOI: 10.1021/acscatal.2c04608
99 phosphate binding pocket Y273 AdaV DOI: 10.1021/acscatal.2c04608 DOI: 10.1038/s41467-024-49147-7
- lost halogeantion activity; reduced hydroxylation activity Q203A&V269A&G196T AdaV DOI: 10.1021/acscatal.2c04608
- lost halogeantion activity; reduced hydroxylation activity Q203A&V269A&G196S AdaV DOI: 10.1021/acscatal.2c04608
- abolished halogenation activity R177A AdaV DOI: 10.1021/acscatal.2c04608
- abolished halogenation activity Q198A AdaV DOI: 10.1021/acscatal.2c04608
129 phosphate binding site F303 AdaV DOI: 10.1038/s41467-024-49147-7
20 direct halogenation of a nucleotide H194 AdaV DOI: 10.1021/acscatal.2c04608 DOI: 10.1038/s41467-024-49147-7
97 substrate binding pocket; H-bond with deoxyribose moiety F271 AdaV DOI: 10.1021/acscatal.2c04608 DOI: 10.1038/s41467-024-49147-7
- hydroxylation and chlorination activity; bind Fe2+ to the same extent as the V269A AdaV DOI: 10.1021/acscatal.2c04608
- increased hydroxylation activity to the halogenation activity (compared with the single mutation variants) Q203A&V269A AdaV DOI: 10.1021/acscatal.2c04608
- abolished halogenation activity F271A AdaV DOI: 10.1021/acscatal.2c04608
- reduced activity P106A AdaV DOI: 10.1021/acscatal.2c04608
- reduced activity; retained 38% activity R182A AdaV DOI: 10.1021/acscatal.2c04608
- reduced activity R237A AdaV DOI: 10.1021/acscatal.2c04608
- maintained 5% activity of the WT Y273A AdaV DOI: 10.1021/acscatal.2c04608
- phosphate binding pocket; H-bond network R177 AdaV DOI: 10.1021/acscatal.2c04608 DOI: 10.1038/s41467-024-49147-7
- phosphate-binding site N175 AdaV DOI: 10.1038/s41467-024-49147-7
98 phosphate-binding site A272 AdaV DOI: 10.1038/s41467-024-49147-7
125 phosphate-binding site I299 AdaV DOI: 10.1038/s41467-024-49147-7
- lost halogeantion activity; reduced hydroxylation activity Q203A&V269A&G196D AdaV DOI: 10.1021/acscatal.2c04608
- lost halogeantion activity; reduced hydroxylation activity Q203A&V269A&G196C AdaV DOI: 10.1021/acscatal.2c04608
- lost halogeantion activity; reduced hydroxylation activity Q203A&V269A&G196L AdaV DOI: 10.1021/acscatal.2c04608
78 direct halogenation of a nucleotide H252 AdaV DOI: 10.1021/acscatal.2c04608 DOI: 10.1038/s41467-024-49147-7
- no chlorination activity G196A AdaV DOI: 10.1021/acscatal.2c04608
- no chlorination activity G196D AdaV DOI: 10.1021/acscatal.2c04608
- no hydroxylated product; partially attenuated halogenation activity; bind Fe2+ to the same extent as the G201A AdaV DOI: 10.1021/acscatal.2c04608
- no hydroxylation/halogenation activity G196C AdaV DOI: 10.1021/acscatal.2c04608
- no hydroxylation/halogenation activity G196L AdaV DOI: 10.1021/acscatal.2c04608
- no hydroxylation/halogenation activity G196T AdaV DOI: 10.1021/acscatal.2c04608
- no hydroxylation/halogenation activity G196S AdaV DOI: 10.1021/acscatal.2c04608
- no chlorination activity; O blocks chlorine ligand binding and there is no electron density for the chlorine ligand close to the active pocket G196E AdaV DOI: 10.1021/acscatal.2c04608
- hydroxylation and chlorination activity; bind Fe2+ to the same extent as the Q203A AdaV DOI: 10.1021/acscatal.2c04608
- no hydroxylated product; partially attenuated halogenation activity; bind Fe2+ to the same extent as the N234A AdaV DOI: 10.1021/acscatal.2c04608
- abolished halogenation activity H251F AdaV DOI: doi.10.1002/anie.201914994