Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.605680
Title: Structural and mechanistic aspects of catalytic promiscuity in the alkaline phosphatase superfamily
Author: Jonas, S.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2009
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Abstract:
In this thesis I explored the catalytic promiscuity of two very closely related phosphonate monoester hydrolases from Burkholderia caryophylii (BcPMH) and Rhizobium leguminosarum (RIPMH) which display hydrolytic activities towards six substrates: phosphonate monoester, phosphate monoester, -diester, and –triester, sulfate monoester and sulfonate monoester. This substrate collection encompasses a range of substrate charges between 0 and -2, transition states of a different nature and involves attack at two different reaction centers (P and S). Intrinsic reactivities (half lives) range from 200 days to 105 years under near neutrality. The highest efficiencies are achieved in hydrolyses of phosphonate monoesters and phosphate diesters, the last of which has been suggested as the native function. MALDI-TOF analysis shows that both enzymes employ a formylglycine as a nucleophile, the product of post-translational oxidation of a cysteine, which has so far only been described as the signature residue of sulfatases. The crystal structures of both enzymes show their close relationships to alkaline phosphatase (AP) and arylsulfatases and establish them as new members of the AP superfamily. A spacious open active site allows the enzymes to accommodate substrates of varying sizes, while the metal-activated nucleophile in the mononuclear active site provides a substantial portion of the high reactivity necessary to convert all of the six substrates. PMH encompasses four of the native activities previously observed in the AP superfamily and extends its repertoire by two further activities, one of which, sulfonate monoesterase, has not been observed previously for a natural enzyme. PMH is thus one of the most promiscuous hydrolases described to date. Mutational analysis of the active site of RIPMH in complex with vanadate or the product of the phosphonate monoesterase reaction, phenyl phosphonate, were used to propose a mechanism for the native and additional reactions.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.605680  DOI: Not available
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