2vef: Difference between revisions

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'''DIHYDROPTEROATE SYNTHASE FROM STREPTOCOCCUS PNEUMONIAE'''

Dihydropteroate synthase (DHPS) catalyses an essential step in the biosynthesis of folic acid and is the target for the sulfonamide group of antimicrobial drugs. Here we report two crystal structures of DHPS from the respiratory pathogen Streptococcus pneumoniae: the apoenzyme at 1.8-A resolution and a complex with 6-hydroxymethyl-7,8-dihydropterin monophosphate at 2.4-A resolution. The enzyme forms a alpha/beta barrel structure, with a highly conserved binding pocket for recognition of the pterin substrate, 6-hydroxymethyl-7,8-dihydropterin pyrophosphate (DHPPP). There is a fixed order of substrate binding: DHPPP binds first, followed by the second substrate, para-aminobenzoic acid (pABA). Binding of pyrophosphate also allows the enzyme to recognise pABA or sulfonamide drugs, which act as pABA analogues. Using equilibrium and pre-steady state kinetic fluorescence measurements, we show that the on-rate for DHPPP binding to the enzyme is relatively slow (2.6 x 105 M-1s-1) and propose that binding of this substrate induces a large scale movement of the second loop in the enzyme structure, to participate in formation of the pABA binding site. Two mutations, which confer resistance to sulfonamide drugs, do not affect DHPPP binding but have a substantial effect on pABA and sulfonamide recognition. The results show that binding of DHPPP and pABA are separate, distinguishable events in the reaction cycle, and that mutations which confer resistance to sulfonamide drugs act exclusively on the second step in the binding process.

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