Amino Acid Substitutions in a Variant of IMP-1 Metallo-β-Lactamase

DOI: 10.1128/AAC.44.8.2023-2027.2000

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Fig. 1.
Construction of the hybrid and parent metallo-β-lactamase genes. The procedure is explained in Materials and Methods. The horizontal single lines represent multicloning regions of the vector plasmids flanking the inserts. TheEcoRI (E) and HindIII (H) sites located in the multicloning regions are indicated above the lines. Inserts are shown by boxes, in which the regions of metallo-β-lactamase genes are shaded in black. The sites of the restriction enzymes are indicated above the boxes. Abbreviations not used in the text are B (BamHI) and X (XbaI). The sites indicated under the boxes are P (for promoter) (14), G (for guanine), and A (for adenine).

Table 1.

Resistance levels conferred by the parent and hybrid metallo-β-lactamases^a

Plasmid	β-Lactamase	Amino acid residue at position:		MIC (μg/ml) of^b:
Plasmid	β-Lactamase	87	196	LOR	CEF	CTX	CAZ	AMP	IMP	ATM
pMS401	IMP-3	Gly	Gly	64	512	64	64	8	1	<0.5
pMS402	Hybrid	Glu	Gly	64	512	64	64	8	1	<0.5
pMS502	Hybrid	Gly	Ser	32	128	32	64	64	2	<0.5
pMS501	IMP-1	Glu	Ser	32	128	32	64	64	2	<0.5
pHSG398 Δ Hd				2	8	<0.5	<0.5	4	<0.5	<0.5
None				2	8	<0.5	<0.5	4	<0.5	<0.5

↵a Host strain: E. coli K-12 JM83.
↵b LOR, cephaloridine; CEF, cephalothin; CTX, cefotaxime; CAZ, ceftazidime; AMP, ampicillin; IMP, imipenem; ATM, aztreonam.

Table 2.

Kinetic parameters for hydrolysis of various β-lactam antibiotics by the hybrid and parent metallo-β-lactamases

Plasmid	Enzyme	Amino acid at position:		Kinetic parameter	Antibiotic^a
Plasmid	Enzyme	87	196	Kinetic parameter	LOR	CEF	CTX	CAZ	PEN	AMP	IMP
pMS401	IMP-3	Gly	Gly	k _cat(s⁻¹)	221 ± 15^b	223 ± 22	40.1 ± 2.6	4.5 ± 0.4^b	14.3 ± 0.8^b	7.4 ± 1.2^b	92.3 ± 2^b
				K_m (μM)	248 ± 39^b	9.9 ± 1.3	3.1 ± 0.3	128 ± 17^b	370 ± 59^b	464 ± 145^b	1140 ± 40^b
				k _cat/K_m (μM⁻¹s⁻¹)	0.89	22.5	12.9	0.035	0.039	0.016	0.08
pMS402	Hybrid	Glu	Gly	k _cat(s⁻¹)	268 ± 5^b	384 ± 11	54.8 ± 5.3	3.6 ± 0.2^b	21.6 ± 1.0^b	11.8 ± 1.4^b	132 ± 8^b
				K_m (μM)	215 ± 17^b	9.8 ± 0.3	3.0 ± 0.3	118 ± 19^b	464 ± 60^b	437 ± 105^b	1280 ± 120^b
				k _cat/K_m (μM⁻¹s⁻¹)	1.25	39.2	18.3	0.031	0.047	0.027	0.10
pMS502	Hybrid	Gly	Ser	k _cat(s⁻¹)	38.4 ± 1.2	43.2 ± 1.7	16.5 ± 1.3	11.3 ± 0.3	282 ± 19	87.0 ± 2.7	90.8 ± 5.3
				K_m (μM)	7.1 ± 0.1	2.3 ± 0.1	1.3 ± 0.2	31.8 ± 1.5	216 ± 13	110 ± 4.9	33.2 ± 1.9
				k _cat/K_m (μM⁻¹s⁻¹)	5.4	18.8	12.7	0.36	1.3	0.8	2.7
pMS501	IMP-1	Glu	Ser	k _cat(s⁻¹)	62.0 ± 3.0	65.0 ± 2.9	22.5 ± 1.9	16.3 ± 0.9	461 ± 17	162 ± 15	127 ± 11
				K_m (μM)	7.2 ± 0.4	2.0 ± 0.1	1.4 ± 0.1	46.0 ± 1.4	241 ± 10	140 ± 9	30.0 ± 3.9
				k _cat/K_m (μM⁻¹s⁻¹)	8.6	32.5	16.1	0.35	1.9	1.2	4.2