Structural Studies of the Methylosinus trichosporium OB3b Soluble Methane Monooxygenase Hydroxylase and Regulatory Component Complex Reveal a Transient Substrate Tunnel

Abstract

The metalloenzyme soluble methane monooxygenase (sMMO) consists of hydroxylase (sMMOH), regulatory (MMOB), and reductase components. When sMMOH forms a complex with MMOB, the rate constants are greatly increased for the sequential access of O2, protons, and CH4 to an oxygen-bridged diferrous metal cluster located in the buried active site. Here, we report high-resolution X-ray crystal structures of the diferric and diferrous states of both sMMOH and the sMMOH:MMOB complex using the components from Methylosinus trichosporium OB3b. These structures are analyzed for O2 access routes enhanced when the complex forms. Previously reported, lower-resolution structures of the sMMOH:MMOB complex from the sMMO of Methylococcus capsulatus Bath revealed a series of cavities through sMMOH postulated to serve as the O2 conduit. This potential role is evaluated in greater detail using the current structures. Additionally, a search for other potential O2 conduits in the M. trichosporium OB3b sMMOH:MMOB complex revealed a narrow molecular tunnel, termed the W308-tunnel. This tunnel is sized appropriately for O2 and traverses the sMMOH–MMOB interface before accessing the active site. The kinetics of reaction of O2 with the diferrous sMMOH:MMOB complex in solution show that use of the MMOB V41R variant decreases the rate constant for O2 binding >25000-fold without altering the component affinity. The location of Val41 near the entrance to the W308-tunnel is consistent with the tunnel serving as the primary route for the transfer of O2 into the active site. Accordingly, the crystal structures show that formation of the diferrous sMMOH:MMOB complex restricts access through the chain of cavities while opening the W308-tunnel.

Soluble Methane Monooxygenase Component Interactions Monitored by 19F NMR

“… , Binding of MMOB effects a 1000-fold increase in the rate constant for the O 2 binding to the diiron cluster to form the first spectroscopically distinct intermediate of the reaction cycle, termed P* . ,− Recent structural studies indicate that one cause of the decreased rate of O 2 binding in the sMMOH active site in the absence of MMOB is the near closure of the molecular tunnel that mediates the transit of O 2 from the solvent . This bottleneck is relieved by conformational changes in both MMOB and sMMOH red when the sMMOH red :MMOB complex forms. , A second cause of the low reactivity of O 2 with sMMOH red is the position of the Glu209 ligand to the diiron cluster, which blocks the approach to the open iron coordination site . An angle change of this residue in the sMMOH red :MMOB complex exposes the site for O 2 binding .…”

New Trends in Bioremediation Technologies Toward Environment-Friendly Society: A Mini-Review

“…Similarly, Culpepper et al have characterized the molecular structure of pMMO (Culpepper and Rosenzweig, 2012 ), and Rigoldi et al have shown improved catalytic efficiency of pMMO improved by widening the diameter of the active site (Rigoldi et al, 2020 ). A recent study on sMMO showed an essential role of O 2 transport passage to the active site termed as W308-tunnel (Jones et al, 2020 ). Thus, reengineering sMMO might improve the enzymatic efficiency.…”

Design of a Microbial Remediation Inoculation Program for Petroleum Hydrocarbon Contaminated Sites Based on Degradation Pathways

“…BR (PDB ID: 4USQ) [34], flavin-dependent monooxygenase of Nitrincola lacisaponensis (PDB ID: 6HNS) [35], bacterial cytochrome P450 enzyme of Bacillus megaterium (PDB ID: 1SMJ) [36], and cytochrome P450 enzyme of Streptomyces avermitilis (PDB ID: 3E5K) [37] were selected. The hydroxylation pathway was conducted using five kinds of hydroxylases: methane monooxygenase hydroxylase of Methylococcus capsulatus (PDB ID: 1FZI) [38], methane monooxygenase hydroxylase of Methylosinus trichosporium (PDB ID: 1MHZ) [39], longchain alkane monooxygenase of Geobacillus thermodenitrificans (PDB ID: 3B9N) [40], methane monooxygenase hydroxylase of Methylosinus sporium (PDB ID: 6D7K) [41], and methane monooxygenase hydroxylase of Methylosinus trichosporium OB3b (PDB ID: 6VK6) [42]. The secondary single oxygenation pathway was carried out in the presence of the cytochrome P450 enzyme of Streptomyces avermitilis (PDB ID: 3E5K) [37], alcohol dehydrogenase of Escherichia coli (PDB ID: 7BU3) [43], alcohol dehydrogenase of Saccharomyces cerevisiae (PDB ID: 1Q1N) [44], aldehyde dehydrogenase of Staphylococcus aureus (PDB ID: 6K10) [45], and aldehyde dehydrogenase of Pseudomonas aeruginosa (PDB ID: 4CAZ) [46].…”

“…The secondary single oxygenation pathway was carried out in the presence of the cytochrome P450 enzyme of Streptomyces avermitilis (PDB ID: 3E5K) [37], alcohol dehydrogenase of Escherichia coli (PDB ID: 7BU3) [43], alcohol dehydrogenase of Saccharomyces cerevisiae (PDB ID: 1Q1N) [44], aldehyde dehydrogenase of Staphylococcus aureus (PDB ID: 6K10) [45], and aldehyde dehydrogenase of Pseudomonas aeruginosa (PDB ID: 4CAZ) [46]. The secondary hydroxylation pathway was conducted in the presence of the methane monooxygenase hydroxylase of Methylococcus capsulatus (PDB ID: 1FZI) [38], methane monooxygenase hydroxylase of Methylosinus sporium (PDB ID: 6D7K) [41], and methane monooxygenase hydroxylase of Methylosinus trichosporium OB3b (PDB ID: 6VK6) [42].…”

“…The degradation pathways of benzene and the types of degradation enzymes used were different from those used for the degradation of the other three petroleum hydrocarbon pollutants. The degraded proteins selected in each step of the benzene degradation path were identified: the hydroxylation pathway involved the participation of the methane monooxygenase hydroxylase of Methylococcus capsulatus (PDB ID: 1FZI) [38], methane monooxygenase hydroxylase of Methylosinus trichosporium (PDB ID: 1MHZ) [39], longchain alkane monooxygenase of Geobacillus thermodenitrificans (PDB ID: 3B9N) [40], methane monooxygenase hydroxylase of Methylosinus sporium (PDB ID: 6D7K) [41], and methane monooxygenase hydroxylase of Methylosinus trichosporium OB3b (PDB ID: 6VK6) [42]. The secondary single oxygenation pathway involved the participation of five kinds of dioxygenases: procatechuic acid 2,3-dioxygenase of Brevibacterium fuscum (PDB ID: 2IGA) [47], catechol 1,2-dioxygenase of Acinetobacter baylyi ADP1 (PDB ID: 1DLT) [48], catechin acid 1,2dioxygenase of Pseudomonas putida (PDB ID: 2AZQ) [49], 2,3-dioxygenase of Pseudomonas alkylphenolica (PDB ID: 3HPY) [50], and catechol 1,2-dioxygenase of Acinetobacter radioresistens OB3b (PDB ID: 2XSR) [51].…”