Exploring the role of a conserved class A residue in the Ω-Loop of KPC-2 ß-lactamase: a mechanism for ceftazidime hydrolysis.

Gram-negative bacteria harboring KPC-2, a class A ß-lactamase, are resistant to all ß-lactam antibiotics and pose a major public health threat. Arg-164 is a conserved residue in all class A ß-lactamases and is located in the solvent-exposed Ω-loop of KPC-2. To probe the role of this amino acid in KPC-2, we performed site-saturation mutagenesis. When compared with wild type, 11 of 19 variants at position Arg-164 in KPC-2 conferred increased resistance to the oxyimino-cephalosporin, ceftazidime (minimum inhibitory concentration; 32→128 mg/liter) when expressed in Escherichia coli. Using the R164S variant of KPC-2 as a representative ß-lactamase for more detailed analysis, we observed only a modest 25% increase in k(cat)/K(m) for ceftazidime (0.015→0.019 µm(-1) s(-1)). Employing pre-steady-state kinetics and mass spectrometry, we determined that acylation is rate-limiting for ceftazidime hydrolysis by KPC-2, whereas deacylation is rate-limiting in the R164S variant, leading to accumulation of acyl-enzyme at steady-state. CD spectroscopy revealed that a conformational change occurred in the turnover of ceftazidime by KPC-2, but not the R164S variant, providing evidence for a different form of the enzyme at steady state. Molecular models constructed to explain these findings suggest that ceftazidime adopts a unique conformation, despite preservation of Ω-loop structure. We propose that the R164S substitution in KPC-2 enhances ceftazidime resistance by proceeding through "covalent trapping" of the substrate by a deacylation impaired enzyme with a lower K(m). Future antibiotic design must consider the distinctive behavior of the Ω-loop of KPC-2.

Genome maintenance defects in cultured cells and mice following partial inactivation of the essential cell cycle checkpoint gene Hus1.

Cell cycle checkpoints are evolutionarily conserved signaling pathways that uphold genomic integrity. Complete inactivation of the mouse checkpoint gene Hus1 results in chromosomal instability, genotoxin hypersensitivity, and embryonic lethality. To determine the functional consequences of partial Hus1 impairment, we generated an allelic series in which Hus1 expression was incrementally reduced by combining a hypomorphic Hus1 allele, Hus1(neo), with either wild-type or null (Hus1(Delta1)) alleles. Primary Hus1(neo/Delta1) embryonic fibroblasts exhibited spontaneous chromosomal abnormalities and underwent premature senescence, while higher Hus1 expression in Hus1(neo/neo) cells allowed for normal proliferation. Antioxidant treatment almost fully suppressed premature senescence in Hus1(neo/Delta1) cultures, suggesting a critical role for Hus1 in oxidative stress responses. Treatment of Hus1(neo/neo) and Hus1(neo/Delta1) cells with the DNA adducting agent benzo(a)pyrene dihydrodriol epoxide resulted in a loss of cell viability that was associated with S-phase DNA damage checkpoint failure. Likewise, the DNA polymerase inhibitor aphidicolin triggered increased cell death, chromosomal aberrations, and H2AX phosphorylation, a marker for double-stranded DNA breaks, in Hus1(neo/neo) and Hus1(neo/Delta1) cultures compared to controls. Despite these pronounced genome maintenance defects in cultured Hus1(neo/Delta1) and Hus1(neo/neo) cells, mice of the same genotypes were born at expected frequencies and appeared grossly normal. A significant increase in micronucleus formation was observed in peripheral blood cells from Hus1(neo/Delta1) mice, but reduced Hus1 expression did not cause an elevated predisposition to spontaneous tumor development or accelerate tumorigenesis in p53-deficient mice. These results identify differential effects of altered Hus1 gene dosage on genome maintenance during in vitro culture, genotoxic stress responses, embryonic development, and adult homeostasis.

Conditional inactivation of the mouse Hus1 cell cycle checkpoint gene.

The Hus1 cell cycle checkpoint protein plays a central role in genome maintenance by mediating cellular responses to DNA damage and replication stress. Targeted deletion of mouse Hus1 results in spontaneous chromosomal abnormalities and embryonic lethality. To study the physiological impact of Hus1 deficiency in adult mice, we generated a conditional Hus1 allele, Hus1(flox), in which exons two and three are flanked by loxP sites. Cre-mediated excision of the loxP-flanked region produces Hus1(Delta2,3), which is capable of encoding only 19 of 281 Hus1 amino acids. Germline homozygosity for Hus1(Delta2,3) resulted in mid-gestational embryonic lethality that was indistinguishable from that caused by an established null allele, Hus1(Delta1n). Hus1 was inactivated in adult mice using a transgenic strain in which Cre is sporadically expressed in a variety of tissues from the Hsp70-1 promoter. Conditional Hus1 knockout mice were produced at unexpectedly low frequency and, unlike control animals, demonstrated limited inactivation of the conditional allele, suggesting that Hus1-deficient cells were at a strong selective disadvantage in adult animals. However, viable conditional Hus1 knockout mice consistently showed the greatest degree of Hus1 inactivation specifically in lung and mammary gland, highlighting varying requirements for Hus1 in different tissues. The novel tools described here hold promise for elucidating how the Hus1-dependent checkpoint mechanism contributes to chromosomal stability, DNA damage responses, and tumor suppression in adult mice.