Table 1. CBS Protein Variants Constructed and Analyzed.

Plasmid (pJR #)	Nucleotide Mutation(s)	Substituted Residue	Rosetta (ΔΔG)	Phenotypic Category	400 ng/mL B6	4 ng/mL B6	2 ng/mL B6	1 ng/mL B6
2985	194, A→T	H65L	1.2	Nonfunctional	0	n/d	n/d	n/d
2993	424, AT→GC	I142A	3.7	Nonfunctional	0	n/d	n/d	n/d
2994	425, T→A	I142N	9.7	Nonfunctional	0	n/d	n/d	n/d
2996	460, CT→GC	L154A	1.8	Nonfunctional	0	n/d	n/d	n/d
2997	461, T→C	L154P	6.7	Nonfunctional	0	n/d	n/d	n/d
2998	529, A→G	K177E	1.2	Nonfunctional	0	n/d	n/d	n/d
3001	566, T→C	V189A	3.1	Nonfunctional	0	n/d	n/d	n/d
3002	620, C→G	A207G	2.0	Nonfunctional	0	n/d	n/d	n/d
3003	629, T→A	L210Q	4.2	Nonfunctional	0	n/d	n/d	n/d
3005	659, T→G	L220R	14.4	Nonfunctional	0	n/d	n/d	n/d
3007	684, C→A	N228K	5.5	Nonfunctional	0	n/d	n/d	n/d
3008	718, AT→GC	I240A	2.1	Nonfunctional	0	n/d	n/d	n/d
3012	755, T→C	V252A	2.6	Nonfunctional	0	n/d	n/d	n/d
3013	772, G→C	G258R	13.7	Nonfunctional	0	n/d	n/d	n/d
3018	829, AT→CC	I277P	5.1	Nonfunctional	0	n/d	n/d	n/d
3023	967, T→G	W323G	6.3	Nonfunctional	0	n/d	n/d	n/d
3035	1153, TTC→CAA	F385Q	5.0	Nonfunctional	0	n/d	n/d	n/d
3038	1268, T→C	L423P	n/a	Nonfunctional	0	n/d	n/d	n/d
3040	1370, G→A	G457E	n/a	Nonfunctional	0	n/d	n/d	n/d
2988	353, T→G	V118G	1.9	Sensitive	0.91 ± 0.04	0.81 ± 0.25	0.49 ± 0.23 *	0.39 ± 0.07 **
2991	379, A→G	I127V	1.5	Sensitive	1.22 ± 0.11	1.25 ± 0.07 *	1.35 ± 0.15	0.97 ± 0.08
2995	424, A→G	I142V	1.3	Sensitive	0.66 ± 0.03 **	0.68 ± 0.07 *	0.53 ± 0.07	0.38 ± 0.10
2999	541, CT→GC	L181A	3.1	Sensitive	0.92 ± 0.23	0.91 ± 0.12	0.56 ± 0.11	0.34 ± 0.22 *
3000	562, A→G	I188V	1.0	Sensitive	1.19 ± 0.23	1.41 ± 0.38	1.42 ± 0.53	1.93 ± 0.64 *
3006	674, A→G	N225S	1.4	Sensitive	0.71 ± 0.10 **	0.62 ± 0.11 **	0.35 ± 0.09 **	0.36 ± 0.07 **
3015	799, A→G	K267E	0.9	Sensitive	0.68 ± 0.38	0.67 ± 0.39	0.47 ± 0.28 **	0.26 ± 0.15 **
3020	856, AT→GC	I286A	1.1	Sensitive	0.52 ± 0.10	0.43 ± 0.05 *	0.18 ± 0.05 *	0.13 ± 0.11 *
3021	877, C→G	L293V	1.1	Sensitive	0.57 ± 0.08	0.50 ± 0.05 *	0.17 ± 0.04 *	0.25 ± 0.06 *
3024	1012, CT→GC	L338A	1.7	Sensitive	0.98 ± 0.11	0.93 ± 0.17	0.77 ± 0.13 *	0.74 ± 0.14
3026	1034, T→C	L345P	−1.2	Sensitive	0.57 ± 0.04 **	0.49 ± 0.07 **	0.16 ± 0.03 **	0.20 ± 0.14 **
3027	1061, T→G	V354G	7.5	Sensitive	0.67 ± 0.09 **	0.72 ± 0.05 **	0.48 ± 0.09 **	0.54 ± 0.10 **
3031	1112, T→C	V371A	2.7	Sensitive	1.15 ± 0.29	1.15 ± 0.37	1.32 ± 0.20 *	1.95 ± 0.54
3036	1153, T→C	F385L	5.5	Sensitive	0.85 ± 0.15	0.72 ± 0.05 **	0.37 ± 0.07 **	0.37 ± 0.03 **
2986	250, A→G	I84V	1.0	Benign	0.99 ± 0.05	1.10 ± 0.28	1.13 ± 0.24	1.31 ± 0.34
2990	370, CT→GC	L124A	2.3	Benign	0.83 ± 0.06	1.00 ± 0.06	0.97 ± 0.07	1.12 ± 0.11
2989	370, C→G	L124V	2.1	Benign	0.76 ± 0.16	1.10 ± 0.18	1.09 ± 0.10	1.62 ± 0.45
2992	418, G→A	D140N	3.0	Benign	1.13 ± 0.28	0.93 ± 0.11	0.86 ± 0.08	0.71 ± 0.07
3004	640, A→G	I214V	1.1	Benign	0.89 ± 0.18	1.16 ± 0.19	1.07 ± 0.17	1.11 ± 0.08
3009	718, A→G	I240V	1.0	Benign	0.90 ± 0.24	1.14 ± 0.21	0.90 ± 0.05	1.02 ± 0.33
3010	721, C→G	L241V	1.4	Benign	1.01 ± 0.13	1.03 ± 0.19	0.91 ± 0.19	0.92 ± 0.41
3011	742, CT→GC	L248A	1.2	Benign	0.81 ± 0.08	0.84 ± 0.11	0.73 ± 0.14	0.71 ± 0.08
3014	791, T→C	I264T	3.3	Benign	1.12 ± 0.33	1.13 ± 0.20	1.05 ± 0.33	1.08 ± 0.31
3016	800, A→T	K267M	−1.8	Benign	1.02 ± 0.18	1.07 ± 0.15	1.10 ± 0.21	1.15 ± 0.18
3017	811, A→G	K271E	0.4	Benign	0.87 ± 0.18	0.89 ± 0.28	0.80 ± 0.20	0.87 ± 0.31
3019	839, T→C	V280A	2.5	Benign	1.10 ± 0.30	1.12 ± 0.27	1.13 ± 0.23	1.41 ± 0.21
3022	931, A→G	I311V	0.7	Benign	1.03 ± 0.15	1.33 ± 0.50	1.12 ± 0.44	1.41 ± 0.75
3025	1023, A→T	Q341H	−2.5	Benign	1.09 ± 0.23	0.85 ± 0.20	0.86 ± 0.19	0.86 ± 0.10
3028	1067, T→C	V356A	1.3	Benign	0.98 ± 0.12	1.04 ± 0.06	0.94 ± 0.00	0.92 ± 0.08
3029	1070, C→G	A357G	1.6	Benign	1.09 ± 0.16	1.17 ± 0.23	0.99 ± 0.23	0.77 ± 0.19
3030	1073, T→C	V358A	1.6	Benign	0.92 ± 0.10	0.90 ± 0.05	0.87 ± 0.14	0.73 ± 0.08
3032	1115, T→C	V372A	2.7	Benign	0.75 ± 0.39	0.85 ± 0.48	0.68 ± 0.38	0.68 ± 0.37
3033	1120, CT→GC	L374A	2.3	Benign	1.09 ± 0.10	1.06 ± 0.20	1.17 ± 0.24	1.28 ± 0.22
3034	1147, A→T	T383S	1.1	Benign	0.95 ± 0.15	0.87 ± 0.14	0.70 ± 0.10	0.83 ± 0.22
3037	1223, G→T	W408L	n/a	Benign	1.04 ± 0.03	1.13 ± 0.17	1.19 ± 0.16	1.08 ± 0.10
3039	1298, A→T	H433L	n/a	Benign	0.98 ± 0.22	0.93 ± 0.30	0.83 ± 0.21	0.72 ± 0.13
3041	1468, A→C	I490L	n/a	Benign	0.86 ± 0.49	0.74 ± 0.42	0.74 ± 0.43	0.87 ± 0.49
3042	1646, A→G	D549G	n/a	Benign	0.95 ± 0.13	0.80 ± 0.11	0.69 ± 0.10	0.65 ± 0.12
3043	1675, T→C	Y559H	n/a	Benign	1.36 ± 0.10	1.18 ± 0.32	1.15 ± 0.35	1.07 ± 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.