Characterization of key triacylglycerol biosynthesis processes in rhodococci.

Oleaginous microorganisms have considerable potential for biofuel and commodity chemical production. Under nitrogen-limitation, Rhodococcus jostii RHA1 grown on benzoate, an analog of lignin depolymerization products, accumulated triacylglycerols (TAGs) to 55% of its dry weight during transition to stationary phase, with the predominant fatty acids being C16:0 and C17:0. Transcriptomic analyses of RHA1 grown under conditions of N-limitation and N-excess revealed 1,826 dysregulated genes. Genes whose transcripts were more abundant under N-limitation included those involved in ammonium assimilation, benzoate catabolism, fatty acid biosynthesis and the methylmalonyl-CoA pathway. Of the 16 atf genes potentially encoding diacylglycerol O-acyltransferases, atf8 transcripts were the most abundant during N-limitation (~50-fold more abundant than during N-excess). Consistent with Atf8 being a physiological determinant of TAG accumulation, a Δatf8 mutant accumulated 70% less TAG than wild-type RHA1 while atf8 overexpression increased TAG accumulation 20%. Genes encoding type-2 phosphatidic acid phosphatases were not significantly expressed. By contrast, three genes potentially encoding phosphatases of the haloacid dehalogenase superfamily and that cluster with, or are fused with other Kennedy pathway genes were dysregulated. Overall, these findings advance our understanding of TAG metabolism in mycolic acid-containing bacteria and provide a framework to engineer strains for increased TAG production

Structural Characterization of Pandoraea pnomenusa B-356 Biphenyl Dioxygenase Reveals Features of Potent Polychlorinated Biphenyl-Degrading Enzymes

The oxidative degradation of biphenyl and polychlorinated biphenyls (PCBs) is initiated in Pandoraea pnomenusa B-356 by biphenyl dioxygenase $(BPDO_{B356})$. $BPDO_{B356}$, a heterohexameric $(αβ)_3$ Rieske oxygenase (RO), catalyzes the insertion of dioxygen with stereo- and regioselectivity at the 2,3-carbons of biphenyl, and can transform a broad spectrum of PCB congeners. Here we present the X-ray crystal structures of $BPDO_{B356}$ with and without its substrate biphenyl 1.6-Å resolution for both structures. In both cases, the Fe(II) has five ligands in a square pyramidal configuration: H233 Nε2, H239 Nε2, D386 Oδ1 and Oδ2, and a single water molecule. Analysis of the active sites of $BPDO_{B356}$ and related ROs revealed structural features that likely contribute to the superior PCB-degrading ability of certain BPDOs. First, the active site cavity readily accommodates biphenyl with minimal conformational rearrangement. Second, M231 was predicted to sterically interfere with binding of some PCBs, and substitution of this residue yielded variants that transform 2,2′-dichlorobiphenyl more effectively. Third, in addition to the volume and shape of the active site, residues at the active site entrance also apparently influence substrate preference. Finally, comparison of the conformation of the active site entrance loop among ROs provides a basis for a structure-based classification consistent with a phylogeny derived from amino acid sequence alignments

The impact of nitric oxide toxicity on the evolution of the glutathione transferase superfamily: A proposal for an evolutionary driving force

Background: Why do ancestral GSTs utilize cysteine/serine as catalytic residues, whereas more recently evolved GSTs utilize tyrosine? Results: Only the more recently evolved GSTs display enough affinity to bind and make harmless the toxic DNDGIC (a natur

Studies of a Ring-Cleaving Dioxygenase Illuminate the Role of Cholesterol Metabolism in the Pathogenesis of Mycobacterium tuberculosis

Mycobacterium tuberculosis, the etiological agent of TB, possesses a cholesterol catabolic pathway implicated in pathogenesis. This pathway includes an iron-dependent extradiol dioxygenase, HsaC, that cleaves catechols. Immuno-compromised mice infected with a ΔhsaC mutant of M. tuberculosis H37Rv survived 50% longer than mice infected with the wild-type strain. In guinea pigs, the mutant disseminated more slowly to the spleen, persisted less successfully in the lung, and caused little pathology. These data establish that, while cholesterol metabolism by M. tuberculosis appears to be most important during the chronic stage of infection, it begins much earlier and may contribute to the pathogen's dissemination within the host. Purified HsaC efficiently cleaved the catecholic cholesterol metabolite, DHSA (3,4-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione; kcat/Km = 14.4±0.5 µM−1 s−1), and was inactivated by a halogenated substrate analogue (partition coefficient<50). Remarkably, cholesterol caused loss of viability in the ΔhsaC mutant, consistent with catechol toxicity. Structures of HsaC:DHSA binary complexes at 2.1 Å revealed two catechol-binding modes: bidentate binding to the active site iron, as has been reported in similar enzymes, and, unexpectedly, monodentate binding. The position of the bicyclo-alkanone moiety of DHSA was very similar in the two binding modes, suggesting that this interaction is a determinant in the initial substrate-binding event. These data provide insights into the binding of catechols by extradiol dioxygenases and facilitate inhibitor design

Interaction of cytochrome b₅ and cytochrome c

The interaction and kinetics of electron transfer between cytochrome b₅ and cytochrome c, two well characterised soluble electron transfer proteins, have been investigated by three techniques. First, fluorescence quenching experiments were done with cytochrome b₅ and porphyrin cytochrome c, a fluorescent analogue of cytochrome c. These quenching studies yielded association constants and structural information for the cytochrome b₅ cytochrome c complex. Second, an analysis of the rate of flavin semiquinone reduction of the cytochromes within the cytochrome b₅ cytochrome c complex yielded information about the structure and electrostatics of die complex. Third, the second order rate constant for ferrocytochrome b₅ reduction of ferricytochrome c was determined under a variety of solution conditions by stopped-flow spectroscopy. Particular effort was directed at evaluating the role of the cytochrome bs heme propionates in the interaction and electron transfer between cytochrome b₅ and c through performing each of diese three studies with a derivative of cytochrome b₅ in which die heme propionates had been esterified (referred to as DME-cytochrome b₅). The fluorescence quenching studies on the interaction of cytochrome b₅ and porphyrin cytochrome c and die kinetics of flavin semiquinone reduction of the proteins within the cytochrome b₅ cytochrome c complex provided evidence that esterification of the cytochrome b₅ heme propionates, influences the docking geometry of the two proteins. These findings support the predictions of electrostatic calculations [Mauk, M.R., Mauk, A.G., Weber, P.C. & Matthew, J.B. (1986) Biochemistry, 25, 7085] in two respects. First, esterification of the cytochrome b₅ heme propionates detectably increases the separation between die two heme groups in die cytochrome b₅ cytochrome c complex. Second, die cytochrome c heme group is not as sterically hindered in the DME-cytochiome b₅ cytochrome c complex as in die cytochrome b₅ cytochrome c complex. The stopped-flow studies demonstrate that the bimolecular rate constant for ferrocytochrome b₅ reduction of ferricytochrome c is determined in part by the rate of association of the two proteins. This rate of association is strongly influenced by electrostatic forces. The principal effect of esterification of the cytochrome b₅ heme propionates on the second order rate of electron transfer between cytochromes bs and c is to influence complex formation between these two proteins. The stopped-flow studies further suggest that the reduction potentials of native and DME-cytochromes b₅ are not significantly different when these proteins are bound to cytochrome c. The nature of electron transfer protein-protein interactions and protein-protein electron transfer is discussed with respect to these findings.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat

Characterization of Hybrid Toluate and Benzoate Dioxygenases

Toluate dioxygenase of Pseudomonas putida mt-2 (TADO(mt2)) and benzoate dioxygenase of Acinetobacter calcoaceticus ADP1 (BADO(ADP1)) catalyze the 1,2-dihydroxylation of different ranges of benzoates. The catalytic component of these enzymes is an oxygenase consisting of two subunits. To investigate the structural determinants of substrate specificity in these ring-hydroxylating dioxygenases, hybrid oxygenases consisting of the α subunit of one enzyme and the β subunit of the other were prepared, and their respective specificities were compared to those of the parent enzymes. Reconstituted BADO(ADP1) utilized four of the seven tested benzoates in the following order of apparent specificity: benzoate > 3-methylbenzoate > 3-chlorobenzoate > 2-methylbenzoate. This is a significantly narrower apparent specificity than for TADO(mt2) (3-methylbenzoate > benzoate ∼ 3-chlorobenzoate > 4-methylbenzoate ∼ 4-chlorobenzoate ≫ 2-methylbenzoate ∼ 2-chlorobenzoate [Y. Ge, F. H. Vaillancourt, N. Y. Agar, and L. D. Eltis, J. Bacteriol. 184:4096-4103, 2002]). The apparent substrate specificity of the α(B)β(T) hybrid oxygenase for these benzoates corresponded to that of BADO(ADP1), the parent from which the α subunit originated. In contrast, the apparent substrate specificity of the α(T)β(B) hybrid oxygenase differed slightly from that of TADO(mt2) (3-chlorobenzoate > 3-methylbenzoate > benzoate ∼ 4-methylbenzoate > 4-chlorobenzoate > 2-methylbenzoate > 2-chlorobenzoate). Moreover, the α(T)β(B) hybrid catalyzed the 1,6-dihydroxylation of 2-methylbenzoate, not the 1,2-dihydroxylation catalyzed by the TADO(mt2) parent. Finally, the turnover of this ortho-substituted benzoate was much better coupled to O(2) utilization in the hybrid than in the parent. Overall, these results support the notion that the α subunit harbors the principal determinants of specificity in ring-hydroxylating dioxygenases. However, they also demonstrate that the β subunit contributes significantly to the enzyme's function

The Comparative Abilities of a Small Laccase and a Dye-Decoloring Peroxidase From the Same Bacterium to Transform Natural and Technical Lignins

Funding Information: This work was supported by the Government of Ontario for the project “Forest FAB: Applied Genomics for Functionalized Fibre and Biochemicals” (grant number ORF-RE-05-005), the Natural Sciences and Engineering Research Council (NSERC) of Canada for the Strategic Network Grant “Industrial Biocatalysis Network,” and Genome Canada for the LSARP project “SYNBIOMICS – Functional genomics and techno-economic models for advanced biopolymer synthesis” (grant number 10405) to ERM as well as NSERC Discovery Grant 171359 to LDE. LDE is the recipient of a Tier 1 Canada Research Chair in Microbial Catabolism and Biocatalysis. Publisher Copyright: © Copyright © 2021 Vuong, Singh, Eltis and Master.The relative ability of the small laccase (sLac) and dye-decoloring peroxidase (DyP2) from Amycolatopsis sp. 75iv2 to transform a variety of lignins was investigated using time-of-flight secondary ion mass spectrometry (ToF-SIMS). The enzymes modified organosolv hardwood lignin to different extents even in the absence of an added mediator. More particularly, sLac decreased the lignin modification metric S (S-lignin)/Ar (total aromatics) by 58% over 16h, while DyP2 lowered this ratio by 31% in the absence of exogenous H2O2. When used on their own, both sLac and DyP2 also modified native lignin present in aspen wood powder, albeit to lesser extents than in the organosolv lignin. The addition of ABTS for sLac and Mn2+ as well as H2O2 for DyP2 led to increased lignin modification in aspen wood powder as reflected by a decrease in the G/Ar metric by up to a further 13%. This highlights the importance of exogenous mediators for transforming lignin within its native matrix. Furthermore, the addition of ABTS reduced the selectivity of sLac for S-lignin over G-lignin, indicating that the mediator also altered the product profiles. Finally, when sLac was included in reactions containing DyP2, in part to generate H2O2in situ, the relative abundance of lignin products differed from individual enzymatic treatments. Overall, these results identify possible routes to tuning lignin modification or delignification through choice of enzyme and mediator. Moreover, the current study expands the application of ToF-SIMS to evaluating enzyme action on technical lignins, which can accelerate the discovery and engineering of industrially relevant enzymes for lignin valorization.Peer reviewe