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Mechanism of 4-(β-D-Ribofuranosyl)aminobenzene 5′-Phosphate Synthase, a Key Enzyme in the Methanopterin Biosynthetic Pathway

The first committed step in methanopterin biosynthesis is catalyzed by 4-(β- d -ribofuranosyl)aminobenzene 5′-phosphate (RFA-P) synthase. Unlike all known phosphoribosyltransferases, β-RFA-P synthase catalyzes the unique formation of a C-riboside instead of an N-riboside in the condensation of p...

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Bibliographic Details
Published in:The Journal of biological chemistry 2004-09, Vol.279 (38), p.39389
Main Authors: Razvan V. Dumitru, Stephen W. Ragsdale
Format: Article
Language:English
Online Access:Get full text
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Summary:The first committed step in methanopterin biosynthesis is catalyzed by 4-(β- d -ribofuranosyl)aminobenzene 5′-phosphate (RFA-P) synthase. Unlike all known phosphoribosyltransferases, β-RFA-P synthase catalyzes the unique formation of a C-riboside instead of an N-riboside in the condensation of p -aminobenzoic acid ( p ABA) and 5-phospho-α- d -ribosyl-1-pyrophosphate (PRPP) to produce 4-(β- d -ribofuranosyl)aminobenzene 5′-phosphate (β-RFA-P), CO 2 , and inorganic pyrophosphate (PP i ). Here we report the successful cloning, active overexpression in Escherichia coli , and purification of this homodimeric enzyme containing two 36.2-kDa subunits from the methanogen Methanococcus jannaschii . Steady-state initial velocity and product inhibition kinetic studies indicate an ordered Bi-Ter mechanism involving binding of PRPP, then p ABA, followed by release of the products CO 2 , then β-RFA-P, and finally PP. The Michaelis parameters are as follows: K m p ABA, 0.15 m m ; K m PRPP, 1.50 m m ; V max , 375 nmol/min/mg; k cat , 0.23 s –1 . CO 2 showed uncompetitive inhibition, K i = 0.990 m m , under varied PRPP and saturated p ABA, and a mixed type of inhibition, K 1 = 1.40 m m and K = 3.800 m m , under varied p ABA and saturated PRPP. RFA-P showed uncompetitive inhibition, K i = 0.210 m m , under varied PRPP and saturated p ABA, and again uncompetitive, K i = 0.300 m m , under saturated PRPP and varied p ABA. PP i exhibits competitive inhibition, K i = 0.320 m m , under varied PRPP and saturated p ABA, and a mixed type of inhibition, K 1 = 0.60 m m and K 2 = 1.900 m m , under saturated PRPP and varied p ABA. Synthase lacks any chromogenic cofactor, and the presence of pyridoxal phosphate and the mechanistically related pyruvoyl cofactors has been strictly excluded.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M406442200