Table 1.

Characteristics of the 21 previously identified codons demonstrated to be important for β-gal function through site-directed mutagenesis or protein structure analyses

Domain	Nucleotide range	Codon	NGS missense mutationsa	Sanger missense mutationsb	Total missense	Sequence context	Reported mutation	Reported mutation an SNV? (Y or N)c	Brief Description of (proposed) mutational effects	Reference
1	304–306	Asn102	2	0	2	accAACgtg	Asn → Ala	N	Alters binding affinity to substrate	Wheatley et al.10
1	601–603	Asp201	28	8	36	cagGATatg	Asp → Glu, Asn, Phe	Y	Alters binding affinity to substrate	Xu et al.69
3	1069–1071	His357	4	1	5	cgtCACgag	His → Asp, Phe, Leu, Asn	Y	Destabilizes transition state	Roth et al.66
3	1171–1173	His391	17	6	23	tcgCATtat	His → Glu, Phe, Lys	N	Destabilizes transition state	Huber et al.68
3	1246–1248	Glu416	10	7	17	attGAAacc	Glu → Gln, Val	Y	Alters binding affinity to substrate	Roth et al.64
3	1252–1254	His418	8	3	11	accCACggc	His → Asn, Glu, Phe	Y	Distorts Gly794, alters binding affinity to substrate	Wheatley et al.7, Wheatley et al.10, Roth et al.60, Juers et al.70
3	1378–1380	Asn460	12	6	18	gggAATgaa	Asn → Asp, Thr, Ser	Y	Destabilizes transition state	Wheatley et al.73
3	1381–1383	Glu461	13	4	17	aatGAAtca	Glu → His, Asp, Gly, Gln, His, Lys	Y	Destabilizes transition state; alters binding affinity to substrate	Cupples et al.58, Edwards et al.59, Martinez-Bilbao et al.62, Richard et al.63
3	1507–1509	Tyr503	24	2	26	atgTACgcg	Tyr → Phe, His, Cys, Lys	Y	Reduction in enzyme catalytic activity	Ring et al.34, Ring et al.57, Edwards et al.59, Penner et al.67
3	1549–1551	Lys517	0	0	0	ccgAAAtgg	Lys → Ala	N	Alters binding affinity to substrate	Wheatley et al.10
3	1609–1611	Glu537	37	52	89	tgcGAAtac	Glu → Gln, Asp, Val	Y	Inhibits covalent binding to galactosyl moiety of substrate; reduction in enzyme catalytic activity	Juers et al.32, Yuan et al.61
3	1618–1620	His540	59	8	67	gccCACgcg	His → Glu, Phe, Asn	Y	Alters binding affinity to substrate, destabilizes transition state	Roth et al.65
3	1624–1626	Met542	2	1	3	gcgATGggt	Met → Ala	N	Alters stability of 794–804 loop that open and closes active site	Dugdale et al.71
3	1702–1704	Trp568	47	7	54	gtcTGGgac	–	–	Distorts Gly794	Wheatley et al.7
3	1795–1797	Arg599	3	0	3	gatCGCcag	Arg → Ala	N	Alters stability of 794–804 loop that open and closes active site	Dugdale et al.72
3	1801–1803	Phe601	0	0	0	cagTTCtgt	Phe → Ala	N	Alters stability of 794–804 loop that open and closes active site	Juers et al.32
4	2380–2382	Gly794	0	0	0	attGGCgta	Gly → Ala	Y	Alters binding affinity to substrate	Wheatley et al.10, Juers et al.80
4	2386–2388	Ser796	0	1	1	gtaAGTgaa	Ser → Ala, Thr, Asp	N	Alters binding affinity to substrate	Wheatley et al.10, Jancewicz et al.81
4	2389–2391	Glu797	0	1	1	agtGAAgcg	Glu → Ala, Leu	Y	Alters binding affinity to substrate	Wheatley et al.10, Sutendra et al.82
4	2422–2424	Glu808	0	0	0	gtcGAAcgc	–	–	Alters stability of 794–804 loop that open and closes active site	Jancewicz et al.81
4	2995–2997	Trp999	1	0	1	tccTGGagc	Trp → Leu, Phe, Gly, Tyr	Y	Alters binding affinity to substrate	Wheatley et al.10, Huber et al.83

^aBeal et al. 2020.

^bHistorical data curated in this study.

^cY designates that an SNV can lead to the reported mutation in the lacZ sequence, and N designates that it takes multiple mutations to get the amino acid change. For example, Lys → Ala requires a sequence change of AAA to GCA.