The roles of electrostatic charge pairing in the interaction between cytochrome P-450 and NADPH-cytochrome P-450 reductase

The cytochrome P450 enzyme catalysis requires two electrons transferred from NADPH-cytochrome P450 reductase (reductase) to P450. Electrostatic charge-pairing has been proposed to be one of the major forces in the interaction between P450 and reductase. In order to obtain further insight into the molecular basis for the protein interaction, I used two methods, chemical modification and specific anti-peptide antibodies, to study the involvement and importance of charged amino acid residues. Acetylation of lysine residues of P450c and P450b by acetic anhydride dramatically inhibited the reductase-supported P450c-dependent ethoxycoumarin hydroxylation activity, but P450 activity supported by cumene hydroperoxide is relatively unchanged. The modification of lysine residues of P450c and P450b did not grossly disturb the protein conformation as revealed by several spectral studies. This differential effect of lysine modification on the P450 activity in the system reconstituted with reductase versus the system supported by cumene hydroperoxide suggested an important role for P450 lysine residues in the interaction with reductase. Using $\rm\sp{14}C$-acetic anhydride, P450 lysine residues were labelled and further identified on P450c and P450b. Those lysine residues are at position 97, 271, 279, and 407 for P450c, and 251, 384, 422, 433, and 473 for P450b. Alignment of those identified lysine residues on P450c and P450b with amino acid residues identified in other studies indicated those residues reside in three major sequence areas. Modification of arginine residues of P450b by phenylglyoxal and 2, 3-butanedione have no significant effect on P450 activity either supported by NADPH and reductase or supported by cumene hydroperoxide. Further studies using $\rm\sp{14}C$-phenylglyoxal reveals that no incorporation of phenylglyoxal into P450b was found. These results demonstrated a predominant role of lysine residues of P450 in the electrostatic interaction with reductase. To understand the protein binding sites on each of P450 and reductase, I generated three anti-peptide antibodies against regions on reductase and five anti-peptide antibodies against five putative reductase binding sites on P450c. These anti-peptide antibodies were affinity purified and characterized on ELISA and by Western blot analysis. Inhibition experiments using these antibodies demonstrated that regions 109-120 and 204-220 of reductase are probably the two major binding sites for P450. The association of reductase with cytochromes P450 and cytochrome c may rely on different mechanisms. The data from experiments using anti-peptide (P450c) antibodies supports the important role of P450c lysine residues 271/279 and 458/460 in the interaction with reductase. ^

The molecular basis for beta-lactamase gene expression in B. anthracis, B. cereus and B. thuringiensis

The susceptibility of most Bacillus anthracis strains to β-lactam antibiotics is intriguing considering that the B. anthracis genome harbors two β-lactamase genes, bla1 and bla2, and closely-related species, Bacillus cereus and Bacillus thuringiensis, typically produce β-lactamases. This work demonstrates that B. anthracis bla expression is affected by two genes, sigP and rsp, predicted to encode an extracytoplasmic function sigma factor and an antisigma factor, respectively. Deletion of the sigP/rsp locus abolished bla expression in a penicillin-resistant clinical isolate and had no effect on bla expression in a prototypical penicillin-susceptible strain. Complementation with sigP/rsp from the penicillin-resistant strain, but not the penicillin-susceptible strain, conferred β-lactamase activity upon both mutants. These results are attributed to a nucleotide deletion near the 5' end of rsp in the penicillin-resistant strain that is predicted to result in a nonfunctional protein. B. cereus and B. thuringiensis sigP and rsp homologues are required for inducible penicillin resistance in those species. Expression of the B. cereus or B. thuringiensis sigP and rsp genes in a B. anthracis sigP/rsp-null mutant confers resistance to β-lactam antibiotics, suggesting that while B. anthracis contains the genes necessary for sensing β-lactam antibiotics, the B. anthracis sigP/rsp gene products are insufficient for bla induction. ^ Because alternative sigma factors recognize unique promoter sequence, direct targets can be elucidated by comparing transcriptional profiling results with an in silico search using the sigma factor binding sequence. Potential σP -10 and -35 promoter elements were identified upstream from bla1 bla2 and sigP. Results obtained from searching the B. anthracis genome with the conserved sequences were evaluated against transcriptional profiling results comparing B. anthracis 32 and an isogenic sigP/rsp -null strain. Results from these analyses indicate that while the absence of the sigP gene significantly affects the transcript levels of 16 genes, only bla1, bla2 and sigP are directly regulated by σP. The genomes of B. cereus and B. thuringiensis strains were also analyzed for the potential σP binding elements. The sequence was located upstream from the sigP and bla genes, and previously unidentified genes predicted to encode a penicillin-binding protein (PBP) and a D-alanyl-D-alanine carboxypeptidase, indicating that the σ P regulon in these species responds to cell-wall stress caused by β-lactam antibiotics. ^ β-lactam antibiotics prevent attachment of new peptidoglycan to the cell wall by blocking the active site of PBPs. A B. cereus and B. thuringiensis pbp-encoding gene located near bla1 contains a potential σP recognition sequence upstream from the annotated translational start. Deletion of this gene abolished β-lactam resistance in both strains. Mutations in the active site of the PBP were detrimental to β-lactam resistance in B. cereus, but not B. thuringiensis, indicating that the transpeptidase activity is only important in B. cereus. I also found that transcript levels of the PBP-encoding gene are not significantly affected by the presence of β-lactam antibiotic. Based on these data I hypothesize that the gene product acts a sensor of β-lactam antibiotic. ^