Functional Characterization of 21 Rare Allelic CYP1A2 Variants Identified in a Population of 4773 Japanese Individuals by Assessing Phenacetin O-Deethylation

Cytochrome P450 1A2 (CYP1A2), which accounts for approximately 13% of the total hepatic cytochrome content, catalyzes the metabolic reactions of approximately 9% of frequently used drugs, including theophylline and olanzapine. Substantial inter-individual differences in enzymatic activity have been observed among patients, which could be caused by genetic polymorphisms. Therefore, we functionally characterized 21 novel CYP1A2 variants identified in 4773 Japanese individuals by determining the kinetic parameters of phenacetin O-deethylation. Our results showed that most of the evaluated variants exhibited decreased or no enzymatic activity, which may be attributed to potential structural alterations. Notably, the Leu98Gln, Gly233Arg, Ser380del Gly454Asp, and Arg457Trp variants did not exhibit quantifiable enzymatic activity. Additionally, three-dimensional (3D) docking analyses were performed to further understand the underlying mechanisms behind variant pharmacokinetics. Our data further suggest that despite mutations occurring on the protein surface, accumulating interactions could result in the impairment of protein function through the destabilization of binding regions and changes in protein folding. Therefore, our findings provide additional information regarding rare CYP1A2 genetic variants and how their underlying effects could clarify discrepancies noted in previous phenotypical studies. This would allow the improvement of personalized therapeutics and highlight the importance of identifying and characterizing rare variants.

Temporal expression ofMycobacterium smegmatisrespiratory terminal oxidases

Terminal oxidases provide the final step in aerobic respiration by reducing oxygen. The mycobacteria possess two terminal oxidases: a cytochrome c aa3type and a quinol bd type. We previously isolated a bd-type oxidase knockout mutant of Mycobacterium smegmatis that allowed for functional analysis of the aa3type without the contribution of bd-type activity. Growth of M. smegmatis LR222 and JAM1 (LR222bd::kan) was monitored and the cytochrome content at different time points examined. No difference in aerobic growth was observed between M. smegmatis LR222 and JAM1. Membranes were obtained from these cultures and the oxidase concentrations were calculated from their spectrum. Although the mutant was producing only one oxidase type, this oxidase did not reach wild-type levels of expression, suggesting an additional mechanism for energizing the membrane. Moreover, the concentration of both oxidases in the wild-type strain dropped when cultures entered stationary phase, which was not the case for the aa3-type oxidase of the mutant strain. This oxidase remained at a constant concentration post mid-log phase. RNase protection assays also demonstrated late growth phase dependent message expression of the bd oxidase and that the subunits I and II genes were cotranscribed as an operon.

OmcF, a Putative c-Type Monoheme Outer Membrane Cytochrome Required for the Expression of Other Outer Membrane Cytochromes in Geobacter sulfurreducens

ABSTRACT Outer membrane cytochromes are often proposed as likely agents for electron transfer to extracellular electron acceptors, such as Fe(III). The omcF gene in the dissimilatory Fe(III)-reducing microorganism Geobacter sulfurreducens is predicted to code for a small outer membrane monoheme c-type cytochrome. An OmcF-deficient strain was constructed, and its ability to reduce and grow on Fe(III) citrate was found to be impaired. Following a prolonged lag phase (150 h), the OmcF-deficient strain developed the ability to grow in Fe(III) citrate medium with doubling times and yields that were ca. 145% and 70% of those of the wild type, respectively. Comparison of the c-type cytochrome contents of outer membrane-enriched fractions prepared from wild-type and OmcF-deficient cultures confirmed the outer membrane association of OmcF and revealed multiple changes in the cytochrome content of the OmcF-deficient strain. These changes included loss of expression of two previously characterized outer membrane cytochromes, OmcB and OmcC, and overexpression of a third previously characterized outer membrane cytochrome, OmcS, during growth on Fe(III) citrate. The omcB and omcC transcripts could not be detected in the OmcF-deficient mutant by either reverse transcriptase PCR or Northern blot analyses. Expression of the omcF gene in trans restored both the capacity of the OmcF-deficient mutant to reduce Fe(III) and wild-type levels of omcB and omcC mRNA and protein. Thus, elimination of OmcF may impair Fe(III) reduction by influencing expression of OmcB, which has previously been demonstrated to play a critical role in Fe(III) reduction.

MtrB Is Required for Proper Incorporation of the Cytochromes OmcA and OmcB into the Outer Membrane of Shewanella putrefaciens MR-1

ABSTRACT When grown under anaerobic conditions, Shewanella putrefaciens MR-1 synthesizes multiple outer membrane (OM) cytochromes, some of which have a role in the use of insoluble electron acceptors (e.g., MnO2) for anaerobic respiration. The cytochromes OmcA and OmcB are localized to the OM and the OM-like intermediate-density membrane (IM) in MR-1. The components necessary for proper localization of these cytochromes to the OM have not been identified. A gene replacement mutant (strain MTRB1) lacking the putative OM protein MtrB was isolated and characterized. The specific cytochrome content of the OM of MTRB1 was only 36% that of MR-1. This was not the result of a general decline in cytochrome content, however, because the cytoplasmic membrane (CM) and soluble fractions were not cytochrome deficient. While OmcA and OmcB were detected in the OM and IM fractions of MTRB1, significant amounts were mislocalized to the CM. OmcA was also detected in the soluble fraction of MTRB1. While OmcA and OmcB in MR-1 fractions were resistant to solubilization with Triton X-100 in the presence of Mg2+, Triton X-100 readily solubilized these proteins from all subcellular fractions of MTRB1. Together, these data suggest that MtrB is required for the proper localization and insertion of OmcA and OmcB into the OM of MR-1. The inability of MTRB1 to properly insert these, and possibly other, proteins into its OM likely contributes to its marked deficiency in manganese(IV) and iron(III) reduction. While the localization of another putative OM cytochrome (MtrF) could not be directly determined, an mtrF gene replacement mutant exhibited wild-types rates of Mn(IV) and Fe(III) reduction. Therefore, even if MtrF were mislocalized in MTRB1, it would not contribute to the loss of metal reduction activity in this strain.

Genetic Complementation of an Outer Membrane Cytochrome omcB Mutant of Shewanella putrefaciens MR-1 Requires omcB Plus Downstream DNA

ABSTRACT Anaerobically grown cells of the metal-reducing bacterium Shewanella putrefaciens MR-1 contain multiple outer membrane (OM) cytochromes. A gene replacement mutant (strain OMCB1) lacking the OM cytochrome OmcB is markedly deficient in the reduction of MnO2 and exhibits reduced rates of Fe(III) reduction. The levels of other OM cytochromes are also decreased in OMCB1. Complementation of OMCB1 with wild-type omcB did not restore any of these defects. However, a 21-kb genomic fragment from MR-1, which included omcB and 19 kb of downstream DNA, fully restored MnO2 and Fe(III) reduction and the full complement of OM cytochromes to OMCB1. A 14.7-kb DNA fragment, including omcB and 12 kb of downstream DNA, provided only a modest increase in MnO2 reduction and OM cytochrome content, but it fully restored Fe(III) citrate reduction and partially restored FeOOH reduction. While omcB mRNA was readily detected in this complement, the OmcB protein was not detected in any cellular compartment. The restoration of Fe(III) reduction despite the absence of OmcB suggests that OmcB itself is not required for Fe(III) reduction. Another OM cytochrome, OmcA, was mislocalized to the cytoplasmic membrane of OMCB1. Only the 21-kb genomic fragment was able to restore proper localization of OmcA to the OM. This 21-kb fragment does not contain omcA, but it does contain several open reading frames (ORFs) downstream from omcB. The most downstream of these ORFs (altA) encodes a putative AraC-like transcriptional regulator. However, a gene replacement mutant of altA resembled the wild type with respect to MnO2 reduction, OM cytochrome content, and the localization of OmcA and OmcB to the OM. Since OMCB1 continues to express genes immediately downstream from omcB, the lack of expression of this downstream DNA does not explain its phenotype or the need for the large complementing fragment. The results suggest that the DNA downstream of omcB must be present in cis in order to restore Fe(III) reduction, MnO2 reduction, OM cytochrome content, and the localization of OmcA and OmcB to the OM.