Mutagenesis of the "leucine gate" to explore the basis of catalytic versatility in soluble methane monooxygenase

Abstract

Soluble methane monooxygenase (sMMO) from methane-oxidizing bacteria is a multicomponent nonheme oxygenase that naturally oxidizes methane to methanol and can also cooxidize a wide range of adventitious substrates, including mono- and diaromatic hydrocarbons. Leucine 110, at the mouth of the active site in the alpha subunit of the hydroxylase component of sMMO, has been suggested to act as a gate to control the access of substrates to the active site. Previous crystallography of the wild-type sMMO has indicated at least two conformations of the enzyme that have the "leucine gate" open to different extents, and mutagenesis of homologous enzymes has indicated a role for this residue in the control of substrate range and regioselectivity with aromatic substrates. By further refinement of the system for homologous expression of sMMO that we developed previously, we have been able to prepare a range of site-directed mutations at position 110 in the alpha subunit of sMMO. All the mutants (with Gly, Cys, Arg, and Tyr, respectively, at this position) showed relaxations of regioselectivity compared to the wild type with monoaromatic substrates and biphenyl, including the appearance of new products arising from hydroxylation at the 2- and 3- positions on the benzene ring. Mutants with the larger Arg and Trp residues at position 110 also showed shifts in regioselectivity during naphthalene hydroxylation from the 2- to the 1- position. No evidence that mutagenesis of Leu 110 could allow very large substrates to enter the active site was found, however, since the mutants (like the wild type) were inactive toward the triaromatic hydrocarbons anthracene and phenanthrene. Thus, our results indicate that the "leucine gate" in sMMO is more important in controlling the precision of regioselectivity than the sizes of substrates that can enter the active site.

FIG. 1.

Construction of the plasmid pMD-Mdel3 and its use for marker exchange deletion of the first five genes of the sMMO operon. In addition to the sMMO-encoding mmoXYBZDC operon, the upstream mmoR and mmoG genes are also indicated. These encode, respectively, a putative σ⁵⁴-dependent transcriptional regulator and molecular chaperon that (while not part of the mature sMMO complex) are needed for expression of active sMMO in vivo (39). PCR primers for amplification of ′mmoC were mmodel-1 (5′-ATA ATA GGA TCC ATC GTC ATC GAG ACC GAG GAC G-3′; BamHI site underlined) and the M13 universal sequencing primer (3′-GTA AAA CGA CGG CCA GT-5′). Deletion of mmoXYBZD and the upstream region of the mmoC gene was effected by introducing pMD-Mdel3 into M. trichosporium by conjugation (25) and selecting exconjugants by using gentamicin. M. trichosporium SMDM, the exconjugant in which the sMMO-encoding operon had been deleted via double homologous recombination, was selected on the basis of its sMMO-negative phenotype as determined by the naphthalene oxidation test (1, 2), together with its Gm^r Kn^S phenotype. The image is not drawn to scale.

FIG. 2.

Stabilities of the colored diazo compounds formed by reaction of tetrazotised o-dianisidine with 1-naphthol (solid line) or 2-naphthol (dotted line).