Isolation and phenotypic characterization of methanol oxidation mutants of the restricted facultative methylotroph Methylophaga marina

Abstract

Methanol oxidation in Mefhyfophuga marina appears to be organized in a way similar to that in other Gramnegative methylotrophs, involving a quinoprotein methanol dehydrogenase (MDH; EC 1.1.99.8) and two soluble cytochromes. The MDH is composed of two different subunits, most probably arranged in an a2P2 structure. The two cytochromes are of the e-type and differ in size (molecular mass 19.5 and 12.5 kDa) and isoelectric point (PI 4.6 and 9.2). The one with the lowest isoelectric point, commonly designated as cytochrome eL, is able to oxidize reduced MDH. Taking advantage of the ability of M. marina, a restricted facultative methylotroph, to utilize fructose as a growth substrate, mutants impaired in methanol utilization were isolated after application of optimal concentrations of ethylmethane sulphonate. Three classes of methanol oxidation mutants were obtained. Class I mutants were affected in a global regulation of the synthesis of both apo-methanol-dehydrogenase and cytochrome cL as well as PQQ (pyrroloquinoline quinone). Class I1 mutants did not produce an active MDH, but instead a comparable amount of a 65 kDa protein was found in the cell-free extract upon SDS-PAGE. This mutant protein was purified and compared to wild-type MDH. It was located in the periplasm, but unlike MDH it was composed of only two identical large subunits. Each of these subunits was able to bind one molecule of PQQ. An antiserum raised against wild-type MDH did not react with the mutant protein. Conversely, an antiserum raised against mutant protein weakly cross-reacted with wild-type MDH, suggesting that the presence of the P-subunit in MDH dramatically changes its immunochemical behaviour. Three of the class I1 mutants did not produce PQQ. In the presence of PQQ, partial revertants able to grow on methanol medium were obtained (class 111). Class 111 mutants produced a stable apo-MDH consisting of a-and P-subunits and showing a normal reaction with the anti-MDH serum. Although this apo-MDH can bind PQQ, enzymic activity was not restored in nitro. This suggests that, in addition to apo-MDH and PQQ, other factors are required for the assembly of an enzymically active MDH.

Interaction of cytochrome c L with methanol dehydrogenase from Methylophaga marina 42:

“…MDH is a 2,7,9-tricarboxy-lH-pyrrolo-[2,3-f] quinoline-4,5-dione (PQQ) dependent enzyme (Duine et al 1986) and three different redox forms, having distinct absorption spectra, have been identified so far (Duine and Frank 1980; Correspondence to: Claude Balny, Institut National de la Sant~ et de la Recherche M6dicale, U128, CNRS, BP 5051, F-34033 Montpellier C6dex 1, France; Tel. Several reports indicate that MDH is composed of two different subunits with M = 60-70 kDa and M= 6-10 kDa, respectively, arranged in an ezB2 structure (Nunn et al 1989;van Spanning et al 1991;Anderson and Lidstrom 1988) and this has also been shown to be the case for the MDH from Methylophaga marina 42 used in the present study (Janvier et al 1992). Several reports indicate that MDH is composed of two different subunits with M = 60-70 kDa and M= 6-10 kDa, respectively, arranged in an ezB2 structure (Nunn et al 1989;van Spanning et al 1991;Anderson and Lidstrom 1988) and this has also been shown to be the case for the MDH from Methylophaga marina 42 used in the present study (Janvier et al 1992).…”

“…MDH and cytochrome c L were prepared from frozen cell paste of Methylophaga marina 42 as previously described (Janvier et al 1992). Wurster's blue was prepared by oxidation of N,N,N',N' tetramethyl-p-phenylene diamine (Michaelis and Gramick 1943).…”

Fourth derivative UV-spectroscopy of proteins under high pressure II. Application to reversible structural changes

“…Ribonuclease-A (EC 3.1.27.5) was from Sigma, St. Louis, methanol dehydrogenase (MEOH-DH) was isolated from Methylophaga marina as described previously (Janvier et al 1992) and adrenodoxin was isolated from bovine adrenal cortex (Suhara et al 1972;Lange et al 1988). The final enzyme concentrations were: adrenodoxin, 50 gM, ribonuclease-A, 73 gM, and methanol dehydrogenase, Figure 1 illustrates the power of fourth derivative spectroscopy: the zero-order UV spectrum only shows a very small undulation around 265 nm, while the fourth derivative spectrum reveals the characteristic spectral features of phenylalanine (see the preceding paper), even in the presence of large absorbance bands originating from the ironsulphur centre and one tyrosine.…”

Activity and Structural Stability of Methanol Dehydrogenase from Methylophaga marina at High Pressure.

“…4) Addition of substrate then occurs to the C(5) carbonyl of PQQ (covalent catalysis) in the oxidised form of MDH, followed by electron transfer [29]. In analogy with Hyphomicrobium MDH, the enzyme from M. marina is specifically oxidised by cytochrome CL [4].…”