Polycyclic Aromatic Hydrocarbon Metabolic Network in Mycobacterium vanbaalenii PYR-1 (cid:1) †

This study investigated a metabolic network (MN) from Mycobacterium vanbaalenii PYR-1 for polycyclic aromatic hydrocarbons (PAHs) from the perspective of structure, behavior, and evolution, in which multilayer omics data are integrated. Initially, we utilized a high-throughput proteomic analysis to assess the protein expression response of M. vanbaalenii PYR-1 to seven different aromatic compounds. A total of 3,431 proteins (57.38% of the genome-predicted proteins) were identiﬁed, which included 160 proteins that seemed to be involved in the degradation of aromatic hydrocarbons. Based on the proteomic data and the previous metabolic, biochemical, physiological, and genomic information, we reconstructed an experiment-based system-level PAH-MN. The structure of PAH-MN, with 183 metabolic compounds and 224 chemical reactions, has a typical scale-free nature. The behavior and evolution of the PAH-MN reveals a hierarchical modularity with funnel effects in structure/function and intimate association with evolutionary modules of the functional modules, which are the ring cleavage process (RCP), side chain process (SCP), and central aromatic process (CAP). The 189 commonly upregulated proteins in all aromatic hydrocarbon treatments provide insights into the global adaptation to facilitate the PAH metabolism. Taken together, the ﬁndings of our study provide the hierarchical viewpoint from genes/proteins/metabolites to the network via functional modules of the PAH-MN equipped with the engineering-driven approaches of modularization and rationalization, which may expand our understanding of the metabolic potential of PYR-1 for bioremediation applications. analyzed by reversed-phase nano-LC-MS/MS (RP nano-LC-MS/MS). RP nano- LC-MS/MS was performed using an Agilent 1200 nano-LC system (Agilent Technologies) coupled on line to a linear ion trap mass spectrometer (LTQ XL; Thermo Reverse-phase separation was performed using a fused silica capillary column (75- (cid:1) m inner (cid:1) m outer Technologies, AZ) was packed with Jupiter 5- (cid:1) m, 18 silica-bonded (Phenomenex, sample was of injected was were eluted at a ﬂow rate of 0.25 (cid:1) l/min using a step linear gradient of 2 to 42% B (0.1% [vol/vol] formic acid in acetonitrile) for and 42 to 98% solvent B for followed by 98% solvent B mass spectrom- eter operated in a data-dependent mode, in which each full MS scan was followed by seven MS/MS scans, where the seven most abundant peptide mo- lecular ions were dynamically selected from the prior MS scan for collision-induced dissociation (CID) using a normalized collision energy of 35%. identiﬁcation and quantitation. were using SEQUEST, running under BioWorks (Rev. 3.3.1 SP1) San CA), against the M. vanbaalenii PYR-1 protein database with the addition of a PhtAc protein sequence to identify peptides. A peptide mass tolerance of cross-cor- relation of false-positive in peptide identiﬁcation to be less 1% by a SEQUEST search of the database comprising forward and reverse protein sequences. peak areas of identiﬁed peptides calculated using Bio-Works’ PepQuan module (Thermo Electron, San Jose, CA), by which peak areas were integrated from their extracted ion chromatograms (XIC) using a minimum intensity threshold of 50,000 counts, mass tolerance of 1.5 Da, and smoothing point identiﬁed summed which are ring cleaved corresponding catechols. ring cleavage compounds (rhombus), SCP, converts them to metabolic intermediates which are ready to enter another round of RCP or CAP and produce active biological precursors. various of steps, to in the functional order of (i) the hydroxylation of side chains of the ring cleavage compounds, (ii) the oxidation of the hydroxylation-generated aldehyde group to a carboxylic acid, and (iii) the decarboxylation of the carboxyl group from the aromatic nucleus. The functional linearity for the rearrangement of metabolites to enter another round of RCP or CAP results in the relatively low in- and out-degrees of the metabolic compounds. more benzene rings may remain, RCP and SCP reiterate until the pathways produce an intermediate to enter CAP. The number of repetitions depends on the number of benzene rings of the HMW PAHs. Diverse PAHs are transformed into the common metabolic intermediate protocatechuate, which then is funneled into CAP. The CAP consists of a series of reactions for the conversion of protocatechuate to small aliphatic compounds, which can directly enter central metabolism. PYR-1, -ketoadipate pathway functions for the completion of the pathway into the cycle intermediates. the