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J. Biol. Chem., Vol. 279, Issue 38, 39389-39395, September 17, 2004

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

Razvan V. Dumitru and Stephen W. Ragsdale

From the Department of Biochemistry, Beadle Center, University of Nebraska, Lincoln, Nebraska 68588-0664

The first committed step in methanopterin biosynthesis is catalyzed by 4-(-D-ribofuranosyl)aminobenzene 5'-phosphate (RFA-P) synthase. Unlike all known phosphoribosyltransferases, -RFA-P synthase catalyzes the unique formation of a C-riboside instead of an N-riboside in the condensation of p-aminobenzoic acid (pABA) and 5-phospho--D-ribosyl-1-pyrophosphate (PRPP) to produce 4-(-D-ribofuranosyl)aminobenzene 5'-phosphate (-RFA-P), CO₂, 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 pABA, followed by release of the products CO₂, then -RFA-P, and finally PP. The Michaelis parameters are as follows: K_mpABA, 0.15 mM; K_mPRPP, 1.50 mM; V_max, 375 nmol/min/mg; k_cat, 0.23 s^–1. CO₂ showed uncompetitive inhibition, K_i = 0.990 mM, under varied PRPP and saturated pABA, and a mixed type of inhibition, K₁ = 1.40 mM and K = 3.800 mM, under varied pABA and saturated PRPP. RFA-P showed uncompetitive inhibition, K_i = 0.210 mM, under varied PRPP and saturated pABA, and again uncompetitive, K_i = 0.300 mM, under saturated PRPP and varied pABA. PP_i exhibits competitive inhibition, K_i = 0.320 mM, under varied PRPP and saturated pABA, and a mixed type of inhibition, K₁ = 0.60 mM and K₂ = 1.900 mM, under saturated PRPP and varied pABA. Synthase lacks any chromogenic cofactor, and the presence of pyridoxal phosphate and the mechanistically related pyruvoyl cofactors has been strictly excluded.

Received for publication, June 9, 2004 , and in revised form, July 12, 2004.

^* This work was supported by National Institutes of Health Grant R41 GM67952-01. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Biochemistry, Beadle Center, University of Nebraska, Lincoln, NE 68588-0664. Tel.: 402-472-2943, Fax: 402-472-7842; E-mail: sragsdale1{at}unl.edu.

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