Enzyme from ocean bacteria reveals a unique catalytic cofactor

A serendipitous finding by Oxford researchers has identified novel features of an ecologically important enzyme.

The alkaline phosphatase PhoX is important for bacterial phosphate acquisition in low phosphate environments such as those found in much of the world’s oceans (pink). The structure of PhoX and of its active site are shown with the cofactor iron atoms orange, calcium ions green, and the oxygen atom red. A phosphate ion bound at the active site is shown in stick representation

The alkaline phosphatase PhoX is important for bacterial phosphate acquisition in low phosphate environments such as those found in much of the world’s oceans (pink). The structure of PhoX and of its active site are shown with the cofactor iron atoms orange, calcium ions green, and the oxygen atom red. A phosphate ion bound at the active site is shown in stick representation†
(Click to enlarge)

The findings, from Professor Ben Berks' group in Biochemistry and Professor Susan Lea's group in the Dunn School of Pathology, are published in a paper in Science (1). Principal researchers on the work are Chien Yong and Pietro Roversi.

The group's work on the alkaline phosphatase PhoX, which has implications across disciplines ranging from chemistry to microbial ecology, has identified a new and unexpected catalytic cofactor. Structural analysis of PhoX suggests a novel mechanism of action not seen in other phosphatases.

Phosphate-containing macromolecules and metabolites are essential components in all living cells. Under conditions of phosphate deficiency, microorganisms produce alkaline phosphatase enzymes to release phosphate from phosphate-containing organic compounds in the environment.

The PhoA family is the best characterised family of alkaline phosphatase and shares sequence similarity to phosphotransfer enzymes found in higher organisms. A completely unrelated type of alkaline phosphatase, PhoX, was discovered more recently. Although PhoX is present in a wide range of microorganisms, little had been known about the enzyme.

'PhoX is found in all three domains of life – in bacteria, eukaryotic algae and archaea – and in bacteria it is as important as PhoA,' explains Professor Berks. 'It has been purified and has been assumed to be a calcium-containing enzyme.' Proteins, he adds, can do limited types of chemistry on their own and so many enzymes rely on metal cofactors – the functional heart of the enzyme - to carry out catalysis.

The discoveries about PhoX came about because the enzyme is a substrate of the protein transporter that Professor Berks' group was studying. On purifying PhoX, they found that it was an unexpected purple colour – suggesting the presence of a transition metal-containing cofactor.

Through their work with departmental colleagues Elspeth Garman and Oliver Zeldin, they have been able to tease apart the metal ion content of the cofactor. They used an approach known as Micro-PIXE (Proton Induced X-ray Emission) pioneered for biological applications by Professor Garman.

'The technique can be used to determine the metal constituents in proteins which are often hard to determine via other methods,' explains Professor Berks. 'It allows researcher to spot a fingerprint of every element in the molecule and so identify all the metals and their relative amounts.'

They found that the catalytic cofactor of PhoX had a composition and structure that had not been seen in other enzyme actives sites. It contains two iron and three calcium ions, and part of the cofactor is an oxo-centred triangular metal cluster - three metal ions with an oxygen atom in the middle.

Biochemical and spectroscopic studies of PhoX were used to complement crystal structure analysis of the enzyme in complex with ligands. Together, they suggest a mechanism in which the active site metal ions bind substrate and the oxygen atom is involved in catalysis.

The mechanism proposed is very unusual, Professor Berks comments. 'Phosphotransfer is one of the most fundamental reactions in biology and so it's surprising to discover that a widely-distributed phosphatase is structurally and mechanistically distinct from currently characterised families.'

'Inorganic chemists have previously synthesised metallo-organic complexes that look a lot like the PhoX cofactor,' adds Pietro Roversi who carried out the X-ray crystallography as a postdoc in Professor Lea's lab. 'These helped us understand the properties of the enzyme active site.'

As well as providing evidence of a novel catalytic mechanism – offering some intriguing chemistry to follow up - the researchers say their findings also have ecological implications.

PhoX is as widely distributed as the PhoA family of alkaline phosphatases amongst ocean microbes and therefore plays a critical role in the global phosphorus cycle. This new study has shown that PhoX requires iron and calcium for activity, in contrast to PhoA which requires zinc and magnesium. Since iron and zinc concentrations in the oceans are often very low, the metal requirements of the two enzymes may limit the ability of microorganisms to obtain phosphate in marine environments.

Reference

  1. A complex iron-calcium cofactor catalyzing phosphotransfer chemistry. Yong, SC, Roversi, P, Lillington, J, Rodriguez, F, Krehenbrink, M, Zeldin, OB, Garman, EF, Lea, SM and Berks, BC. Science (2014) 345:6201 p1170-1173.

See also a 'Perspective' on the paper:
http://www.sciencemag.org/content/345/6201/1120.full

and 'Wild Types', a blog for ASBMB Today:
http://wildtypes.asbmb.org/2014/09/04/surviving-in-the-pacific-ocean-bacterial-style/

 

The map is reproduced with modifications from an image by David Bice at https://www.e-education.psu.edu/earth103/node/694 under a creative commons CC BY-NC-SA 3.0 license

 





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