TABLE 3.

Mutations of Acinetobacter protocatechuate 3,4-dioxygenase and structural consequences

Designation	Amino acid change(s)a	Description of change(s)
A	T12I	Residue in the α subunit along one wall of the active site. Thr12 Oγ1 forms hydrogen bonds with A132O, I135O and G14N. These residues help orient P15 to allow its side chain to interact with the aromatic ring of the substrate.
B	Q122K	Buried side chain forms hydrogen bonds with side chains of R142 and T342. Mutation places two buried positively charged residues in the αβ interface.
C	A123V	Side chain buried under the short helix. Mutation creates van der Waals clashes, which destabilizes the structure of the loop.
D	T141I	Side chain in a group of hydrophobic residues (V143, L170, F185, I187, F197, and F198) in the α subunit. Mutation abolishes hydrogen bonds made by T141 Oγ1 with V196O and R142N as well as creating van der Waals clashes with H140, R142, and/or F197, destabilizing the α subunit.
E	R142C, R142S	R142 makes a buried salt link with D345 and hydrogen bonds with Q122. Mutations bury an unpaired charged side chain (D345) at the αβ interface, destabilizing it.
F	F145V	Side chain buried in a group of hydrophobic residues (L51, P111, I126, P124, V173, A172, and Y183). Cavity produced from mutation destabilizes the α subunit.
G	2×(N152–A153)	Insertion of 2 residues at the N terminus of the short helix near the β subunit may disrupt the helix, destabilizing the α subunit, or may extend the helix, destabilizing the αβ interface.
H	L170R	Side chain buried in a group of hydrophobic residues (V157, L 158, I161, V196, F198, I337, and I339) in the αβ interface. Mutation buries an unpaired charged side chain, destabilizing the interface.
I	S344I	Side chain in a group of hydrophilic residues (Q122, R142, D155, T342, and E345) in the αβ interface. S344 Oγ1 makes hydrogen bonds with Y79 OH and H125 Nɛ2. Mutation abolishes these hydrogen bonds while creating van der Waals clashes.
J	T347I	T347 Oγ1 makes hydrogen bonds with W71 Nɛ1 and L343O in the αβ interface. Mutation abolishes these hydrogen bonds while creating van der Waals clashes.
K	P373L	Side chain in a group of hydrophobic residues (I364, L365, L372, F423, and I442). Replacement with larger side chain creates several van der Waals clashes, destabilizing the β subunit near the local twofold axis.
L	G375	Main chain (φ and Ψ) angles are not allowed for residues other than glycine. Mutation causes refolding of this segment near local twofold axes, destabilizing the β subunit and dodecameric aggregate.
M	A395P	Side chain in a group of hydrophobic residues (V384, V392, V397, I525, F463, and L465) in the β subunit. Mutation of this residue at the N terminus of a β strand produces van der Waals clashes, with the main chain of V392 and K393 destabilizing the β subunit.
N	W400S	Side chain is in a largely hydrophobic environment (V17, L21, L67, F131, and I475) and stacks against the side chain of H462 (iron ligand). W400 Nɛ1 makes a hydrogen bond with a conserved solvent molecule in the postulated oxygen interaction pocket.
O	2×(F423)	F423 is in the loop joining two β strands. Insertion of an additional residue forces reorganization of the loop, destabilizing the β subunit.
P	P458L	Mutation is in a segment near the iron ligands H460 and H462 and adjacent to R457, which is proposed to stabilize the development of a negative charge on the substrate. Mutation creates van der Waals clashes with the main chain of D483-I486, destabilizing the β subunit.
Q	A467V	Mutation creates van der Waals clashes with A365, E468, and/or Q472, destabilizing the β subunit.
X	Δ(N76–T85)	Deletion of residues in a surface loop between β strands. Several contacts with residues (T347–F352) in an adjacent loop. The Y79 side chain is buried in an αβ interface near the site of the S344I and Q122K mutations.
X1	Δ(N76–T85) + L35F	20 Å away from the deletion. Mutation fills a cavity in a group of hydrophobic residues (L90, W92, F351, L396, and F356), stabilizing the αβ interface.
X2	Δ(N76–T85) + N(88a)H	In the same loop as the deletion. Mutation allows more stable loop conformation.
X3	Δ(N76–T85) + N(88a)H + D74N	Mutation is right before deletion. Mutation allows more stable loop conformation.
Y	Δ(A319–P322)	Deletion of residues in a loop that is part of the interaction with other proteins in a dodecameric aggregate. Adjacent to Y324, which makes hydrogen bonds with the carboxyl group of the substrate. Deletion might alter substrate binding.
Y1	Δ(A319–P322) + G13S	Serine side chain makes a hydrogen bond with A132O. Mutation may allow R133 to reposition to form an interaction with the carboxyl group of the substrate.
Y2	Δ(A319–P322) + P317L	On same loop as the deletion. Mutation reduces conformational rigidity of the imino ring, allowing a more stable structure.
Z	Δ(Y437)	At the beginning of a β strand in the β subunit. Aromatic ring interacts with a group of hydrophobic residues (V384, V397, F439, F463, L465, L474, F478, F523, I525, and L527). Preceding Y436 can replace deleted Y437, while region immediately before deletion refolds.
Z1	Δ(Y437) + L465V	Mutation is on a different β strand in the β subunit but is in the group of hydrophobic residues affected by the original mutation and is inserted into the same cavity that includes Y436. Mutation allows a more stable refolding of the loop.

^a2×, tandem duplications; 88a, PcaG residue in Acinetobacter strain ADP1 that aligns with a gap between residues 88 and 89 in P. putida PcaG.