Analysis of genes involved in methanol oxidation in methylobacterium extorquens

Abstract

In methylotrophic bacteria such as Methvlobacterium extorauens methanol is oxidised to Armaldehyde by methanol dehydrogenase (MDH) located in the periplasm. From MDH the electrons are passed along an electron transport chain including 2 cytochromes (cytochrome Cl and cytochrome ch) and finally to a terminal oxidase. The protons released from methanol oxidation form a proton motive force that can be utilised by ATP synthase to produce ATP for the energy requirements of the bacteria. Methanol dehydrogenase has a aiPz configuration. The X-ray structure of this enzyme showed that the active site is found in the a-subunit and contains the prosthetic group pyrrolo- quinoline quinone (PQQ). PQQ is sandwiched between the 'ceiling' of the active site formed by a unique disulphide bridge between adjacent cysteines (Cys 103, Cys 104) and the 'floor' of the active site formed by tryptophan 243. The active site of MDH also contains a calcium atom co-ordinated to both PQQ and 2 protein groups (Glu 177, Asn 261). The mechanism by which MDH catalyses the oxidation of methanol is poorly understood but an active site base (Asp 303) has been implicated in the reaction mechanism. There are at least 32 genes implicated in methanol oxidation. Most of these were identified in two species; Methvlobacterium extorquens and Paracoccus denitrificans. Although the functions of some of these genes have been determined many have not. This thesis describes work carried out to produce a site directed mutation in MDH changing aspartate 303 to a glutamate (D303E). Attempts to make mutations at other amino acids of MDH including active site residues (Arg331, Asn261, Glu 177 and Trp243) and a residue implicated in the interaction of MDH with cytochrome CL (Lys205) were unsuccessful probably due to the high GC content of the mxaP gene. To determine the roles of several methanol oxidation genes found in the large operon (mxaFJGIR(S)ACKLDB) in M.extorquens characterisation of mutants strains was undertaken. This indicated that expression of the a and P subunit genes (mxaF and mxal) is dependent upon each other. Mutations in mxaF. I, R, S and C resulted in strains that could not grow on methanol indicating that these genes have an essential role in methanol oxidation. Mutations in mxaD and in the intergenic region between mxaR and mxaS resulted in strains that could grow on methanol but the MDH activity was lower than wild-type MDH. A double mutant MDH which lacks calcium from the active site fmxaA") and contains an active site base mutation (D303E) was produced with the aim of reconstituting this enzyme with barium to produce an enzyme with potential for studying the reaction mechanism. This enzyme could not be reconstituted with calcium or barium probably because of the altered structure of the active site. Many of the site directed mutants of MDH have resulted in low levels of MDH expression and hence limiting characterisation. A study was carried out on the eSects of growth conditions on the expression of MDH from mutant strains of M.extorquens. This work indicated that changing pH, the concentration of tetracycline, the carbon substrates and the concentration of formaldehyde could not increase the expression of MDH from these mutant strains.

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