Chris Anthony

We published 2 papers together. The first described our new methylotroph and the next slide is the second.













The following slide was included to show what was lost when Boldrewood was demolished. This was my
room, complete with coffee machine and comfort. Murali with back showing was a 4th year medical student solving
a problem with his project and the other is a research student friend Leigh Felton.
Staff rooms were always availble for students to see members of staf.


The foolowing section is describes our work with MDH which was done some years later in collaboration with
Meenakshi Ghosh who came to Oxford from Calcutta to learn how to do X-ray crystallography of proteins.
She had previously had experience only with inorganic crystals. She needed a project so i provided it.


A wonderful collaboration; Meenakshi knew no biochemistry and i knew no crystallography and its interpretation

We made the mistake of relying on an 'expert' to asssy our enzyme for metal ion content and he found none.
After our Japanes friends showed that their Ethanol dehydrogenase contained calcium we found it in MDH.
Our work with a mutant lacking calcium enabled us to reconstitute it with strontium or barium.







The following slide shows the consensus sequence involved in the docking motifs which demonstrated to
Kashahara and Kato that they had a propeller structure which they wrongly interpreted as evidence for a quinoprotind



The mechanism involves an initial proton abstraction (by Asp 303) and a hydride transfer from the
methyl group of methanol. The quinol then passes its electrons one at a time to the specific
cytochrome electron acceptor. After a single electron has been transferred the free radical PQQH is stable,
and MDH is usuall isolated with PQQ in this form (Shown by Frank and Duine)
















3rd Story. Before we knew the structure of MDH we started working on how this enzyme with a novel
prosthetic group interacts with an electron transport chain so that the oxidation of methanol will be
coupled to ATP production.



NOTE: The ATP yield from methanol is very low but this does not matter because almost every atom of carbon
in these bacteria comes by way of this oxidation process so there is ATP to spare. Cell yields are not limited
(as in other bacteria) by ATP but by avaialbility of NADH. Matt Beardmore-Gray calculated that the large
amount of cytochrome and MDH in these bacteria is sufficient to cover the surface in a mono-molecular layer.


The solution of the unusual MDH structure helped us get an X-ray crystallogrqphy unit set up in the Department,
with the appointment of John Cooper and Steve Wood, and they helped solve the structure of the
two methylotroph cytochromes.








NOTE: the immense amount of difficult work on the genetics of our Methylobacterium by Karen Amaratunga and
Simon Dales led them to provide important evidence for all out ideas about structure, mechanism and
electron transport by a programme of site-directed mutagenesis.

Movie of MDH Made by Stuart Findlow, University of Southampton, form the X-ray diffraction coordinates of MDH. If you cannot see movie when you click on arrrow then Go to YouTube clip

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Two other cycles for acetate assimilaion