Table 1 CBS Protein Variants Constructed and Analyzed
Plasmid
(pJR #)Nucleotide Mutation(s)Substituted ResidueRosetta (ΔΔG)Phenotypic Category400 ng/mL B64 ng/mL B62 ng/mL B61 ng/mL B6
2985194, A→TH65L1.2Nonfunctional0n/dn/dn/d
2993424, AT→GCI142A3.7Nonfunctional0n/dn/dn/d
2994425, T→AI142N9.7Nonfunctional0n/dn/dn/d
2996460, CT→GCL154A1.8Nonfunctional0n/dn/dn/d
2997461, T→CL154P6.7Nonfunctional0n/dn/dn/d
2998529, A→GK177E1.2Nonfunctional0n/dn/dn/d
3001566, T→CV189A3.1Nonfunctional0n/dn/dn/d
3002620, C→GA207G2.0Nonfunctional0n/dn/dn/d
3003629, T→AL210Q4.2Nonfunctional0n/dn/dn/d
3005659, T→GL220R14.4Nonfunctional0n/dn/dn/d
3007684, C→AN228K5.5Nonfunctional0n/dn/dn/d
3008718, AT→GCI240A2.1Nonfunctional0n/dn/dn/d
3012755, T→CV252A2.6Nonfunctional0n/dn/dn/d
3013772, G→CG258R13.7Nonfunctional0n/dn/dn/d
3018829, AT→CCI277P5.1Nonfunctional0n/dn/dn/d
3023967, T→GW323G6.3Nonfunctional0n/dn/dn/d
30351153, TTC→CAAF385Q5.0Nonfunctional0n/dn/dn/d
30381268, T→CL423Pn/aNonfunctional0n/dn/dn/d
30401370, G→AG457En/aNonfunctional0n/dn/dn/d
2988353, T→GV118G1.9Sensitive0.91 ± 0.040.81 ± 0.250.49 ± 0.23 *0.39 ± 0.07 **
2991379, A→GI127V1.5Sensitive1.22 ± 0.111.25 ± 0.07 *1.35 ± 0.150.97 ± 0.08
2995424, A→GI142V1.3Sensitive0.66 ± 0.03 **0.68 ± 0.07 *0.53 ± 0.070.38 ± 0.10
2999541, CT→GCL181A3.1Sensitive0.92 ± 0.230.91 ± 0.120.56 ± 0.110.34 ± 0.22 *
3000562, A→GI188V1.0Sensitive1.19 ± 0.231.41 ± 0.381.42 ± 0.531.93 ± 0.64 *
3006674, A→GN225S1.4Sensitive0.71 ± 0.10 **0.62 ± 0.11 **0.35 ± 0.09 **0.36 ± 0.07 **
3015799, A→GK267E0.9Sensitive0.68 ± 0.380.67 ± 0.390.47 ± 0.28 **0.26 ± 0.15 **
3020856, AT→GCI286A1.1Sensitive0.52 ± 0.100.43 ± 0.05 *0.18 ± 0.05 *0.13 ± 0.11 *
3021877, C→GL293V1.1Sensitive0.57 ± 0.080.50 ± 0.05 *0.17 ± 0.04 *0.25 ± 0.06 *
30241012, CT→GCL338A1.7Sensitive0.98 ± 0.110.93 ± 0.170.77 ± 0.13 *0.74 ± 0.14
30261034, T→CL345P−1.2Sensitive0.57 ± 0.04 **0.49 ± 0.07 **0.16 ± 0.03 **0.20 ± 0.14 **
30271061, T→GV354G7.5Sensitive0.67 ± 0.09 **0.72 ± 0.05 **0.48 ± 0.09 **0.54 ± 0.10 **
30311112, T→CV371A2.7Sensitive1.15 ± 0.291.15 ± 0.371.32 ± 0.20 *1.95 ± 0.54
30361153, T→CF385L5.5Sensitive0.85 ± 0.150.72 ± 0.05 **0.37 ± 0.07 **0.37 ± 0.03 **
2986250, A→GI84V1.0Benign0.99 ± 0.051.10 ± 0.281.13 ± 0.241.31 ± 0.34
2990370, CT→GCL124A2.3Benign0.83 ± 0.061.00 ± 0.060.97 ± 0.071.12 ± 0.11
2989370, C→GL124V2.1Benign0.76 ± 0.161.10 ± 0.181.09 ± 0.101.62 ± 0.45
2992418, G→AD140N3.0Benign1.13 ± 0.280.93 ± 0.110.86 ± 0.080.71 ± 0.07
3004640, A→GI214V1.1Benign0.89 ± 0.181.16 ± 0.191.07 ± 0.171.11 ± 0.08
3009718, A→GI240V1.0Benign0.90 ± 0.241.14 ± 0.210.90 ± 0.051.02 ± 0.33
3010721, C→GL241V1.4Benign1.01 ± 0.131.03 ± 0.190.91 ± 0.190.92 ± 0.41
3011742, CT→GCL248A1.2Benign0.81 ± 0.080.84 ± 0.110.73 ± 0.140.71 ± 0.08
3014791, T→CI264T3.3Benign1.12 ± 0.331.13 ± 0.201.05 ± 0.331.08 ± 0.31
3016800, A→TK267M−1.8Benign1.02 ± 0.181.07 ± 0.151.10 ± 0.211.15 ± 0.18
3017811, A→GK271E0.4Benign0.87 ± 0.180.89 ± 0.280.80 ± 0.200.87 ± 0.31
3019839, T→CV280A2.5Benign1.10 ± 0.301.12 ± 0.271.13 ± 0.231.41 ± 0.21
3022931, A→GI311V0.7Benign1.03 ± 0.151.33 ± 0.501.12 ± 0.441.41 ± 0.75
30251023, A→TQ341H−2.5Benign1.09 ± 0.230.85 ± 0.200.86 ± 0.190.86 ± 0.10
30281067, T→CV356A1.3Benign0.98 ± 0.121.04 ± 0.060.94 ± 0.000.92 ± 0.08
30291070, C→GA357G1.6Benign1.09 ± 0.161.17 ± 0.230.99 ± 0.230.77 ± 0.19
30301073, T→CV358A1.6Benign0.92 ± 0.100.90 ± 0.050.87 ± 0.140.73 ± 0.08
30321115, T→CV372A2.7Benign0.75 ± 0.390.85 ± 0.480.68 ± 0.380.68 ± 0.37
30331120, CT→GCL374A2.3Benign1.09 ± 0.101.06 ± 0.201.17 ± 0.241.28 ± 0.22
30341147, A→TT383S1.1Benign0.95 ± 0.150.87 ± 0.140.70 ± 0.100.83 ± 0.22
30371223, G→TW408Ln/aBenign1.04 ± 0.031.13 ± 0.171.19 ± 0.161.08 ± 0.10
30391298, A→TH433Ln/aBenign0.98 ± 0.220.93 ± 0.300.83 ± 0.210.72 ± 0.13
30411468, A→CI490Ln/aBenign0.86 ± 0.490.74 ± 0.420.74 ± 0.430.87 ± 0.49
30421646, A→GD549Gn/aBenign0.95 ± 0.130.80 ± 0.110.69 ± 0.100.65 ± 0.12
30431675, T→CY559Hn/aBenign1.36 ± 0.101.18 ± 0.321.15 ± 0.351.07 ± 0.37
  • The 58 single amino-acid substitutions constructed within the human CBS coding region are listed. The engineered nucleotide change and the resulting change in amino acid residue are shown (reference sequence NM_000071). The calculated change in free energy based on the Rosetta prediction algorithm for each substitution is also listed (ΔΔG, kcal/mol), with the exception of seven substitutions located in the N-terminal portion of the protein that is not part of the structure and therefore no predictions were available (indicated as n/a). All of the protein variants were assigned to one of three phenotypic categories based on empirical measurements of growth: benign, sensitive, or nonfunctional. The relative growth rates of yeast containing 39 single-amino-acid human CBS substitutions with some degree of function are listed. Growth rates are expressed as a ratio relative to the yeast strain expressing the major human CBS allele, grown with the same amount of exogenous pyridoxine supplementation (either 400, 4, 2, or 1 ng/mL pyridoxine), with the SD indicated (determined from at least three independent yeast transformants). Growth rates that differed significantly from the major allele in the same media conditions are indicated either by a single asterisk (P < 0.01) or a double asterisk (P < 0.001). Growth rates that were not determined are indicated (n/d). CBS, cystathionine β-synthase.