TABLE 2.
Summary of mutations identified in genes ddn and fgd1
| nt change by gene | aa change | Frequency | Concn used for selection (μM) | Comment(s) |
|---|---|---|---|---|
| Gene Rv3547/ddn | ||||
| 24ΔT | 8Phe→Phe | 1 | 5 | Single-nucleotide deletion→frameshift |
| 32C→A | 11Ser→STOP | 31 | 1/5 | Early termination codon |
| 65C→T | 22Ser→Leu | 1 | 1 | Decrease in enzyme activity (5) |
| 72/73ΔTA | 24Ile→Ile | 1 | 1 | Double-nucleotide deletion→frameshift |
| 73ΔA | 25-Asn→Ile | 1 | 1 | Single-nucleotide deletion→frameshift |
| 87C →A | 29Tyr→STOP | 1 | 5 | Early termination codon |
| 124C→T | 42Gln→STOP | 1 | 5 | Early termination codon |
| 143T→C | 48Leu→Pro | 1 | 5 | Single-nucleotide substitution |
| 163⌂CGC | 55⌂Arg | 2 | 1 | In-frame 1-aa insertion; loop interacting with polyglutamate tail of F420 |
| 163⌂21 bp | 55⌂7 aa | 1 | 5 | In-frame 7-aa insertion; loop interacting with polyglutamate tail of F420 |
| 232T→C | 78Ser→Pro | 1 | 1 | Single-nucleotide substitution |
| 242G→A | 81Gly→Asp | 1 | 1 | Flanking F420-interacting residue |
| 257C→T | 86Pro→Leu | 1 | 5 | Substitution loses contact with F420 cofactor |
| 262T→C | 88Trp→Arg | 2 | 1 | Loss of H-bond with F420 |
| 290ΔA | 97Lys→Arg | 1 | 5 | Single-nucleotide deletion→frameshift |
| 320T→C | 107Leu→Pro | 1 | 1 | Partially accessible β-strand flanking F420-binding site |
| 361G→A | 121Glu→Lys | 1 | 1 | Single-nucleotide substitution |
| 397T→G | 133Tyr→Asp | 3 | 5 | Localize in PA-824 putative binding site; substitutions of 133Tyr previously shown to abolish activity (5) |
| 409C→T | 137Gln→STOP | 1 | 5 | Nucleotide substitution→early termination codon |
| Total no. of ddn mutants | 53 | |||
| Gene Rv0407/fgd1 | ||||
| 128C→G | 43Pro→Arg | 2 | 5 | Putative glucose-6-phosphate-binding site (16) |
| 212G→A | 71Gly→Asp | 1 | 1 | Flanks F420-binding site |
| 317G→T | 106Gly→Val | 1 | 1 | Putative F420-binding site |
| 336C→A | 112Asn→Lys | 1 | 5 | Putative H-bond with F420 cofactor |
| 146ΔGCCATG | 129Gly→Ala, 130ΔH, 131ΔA | 1 | 5 | Out-of-frame 6-nt (2-aa) deletion |
| 428G→A | 143Trp→STOP | 1 | 1 | Nucleotide substitution→early termination codon |
| 429G→A | 143Trp→STOP | 1 | 5 | Nucleotide substitution→early termination codon |
| 498ΔC | 166Pro→Pro | 1 | 5 | Single-nucleotide deletion→frameshift |
| 506G→C | 169Gly→Ala | 1 | 5 | Loop distant from active site, function unknown |
| 678ΔA | 227Asp→Glu | 1 | 5 | Single-nucleotide insertion→frameshift |
| 688G→A | 230Glu→Lys | 1 | 1 | Putative glucose-6-phosphate-binding site (16) |
| Total no. of fgd1 mutants | 12 |