Crystal Structure at 2.4 Å Resolution of Borrelia burgdorferi Inosine 5‘-Monophosphate Dehydrogenase:  Evidence of a Substrate-Induced Hinged-Lid Motion by Loop 6^,

Abstract

The conversion of inosine 5'-monophosphate (IMP) to xanthosine 5'-monophosphate (XMP) is the committed and rate-limiting reaction in de novo guanine nucleotide biosynthesis. Inosine 5'- monophosphate dehydrogenase (IMPDH) is the enzyme that catalyzes the oxidation of IMP to XMP with the concomitant reduction of nicotinamide adenine dinucleotide (from NAD(+) to NADH). Because of its critical role in purine biosynthesis, IMPDH is a drug design target for anticancer, antiinfective, and immunosuppressive chemotherapy. We have determined the crystal structure of IMPDH from Borrelia burgdorferi, the bacterial spirochete that causes Lyme disease, with a sulfate ion bound in the IMP phosphate binding site. This is the first structure of IMPDH in the absence of substrate or cofactor where the active-site loop (loop 6), which contains the essential catalytic residue Cys 229, is clearly defined in the electron density. We report that a seven residue region of loop 6, including Cys229, is tilted more than 6 A away from its position in substrate- or substrate analogue-bound structures of IMPDH, suggestive of a conformational change. The location of this loop between beta6 and alpha6 links IMPDH to a family of beta/alpha barrel enzymes known to utilize this loop as a functional lid during catalysis. Least-squares minimization, root-mean-square deviation analysis, and inspection of the molecular surface of the loop 6 region in the substrate-free B. burgdorferi IMPDH and XMP-bound Chinese hamster IMPDH show that loop 6 follows a similar pattern of hinged rigid-body motion and indicates that IMPDH may be using loop 6 to bind and sequester substrate and to recruit an essential catalytic residue.

Cryptosporidium parvum IMP Dehydrogenase

“…5). This hypothesis is supported by the varying extents of disorder in this loop in structures of IMPDHs from different sources (39,43,44).…”

“…This loop undergoes hinged rigid body motion but can also assume several conformations (40,43). Clearly, the mobility of this loop will also be a determinant of drug affinity.…”

Crystal Structure of Human Guanosine Monophosphate Reductase 2 (GMPR2) in Complex with GMP

“…The orientation of the proposed active site loop (179-187), which acts as a functional lid during catalysis, is similar to those in some IMPDHs, but subtly different from that in B. burgdorferi IMPDH. [13][14][15][16][17][18] In addition, the location of the b2-sheet is different: it is far away from the active site and may have no contribution to the catalytic activity. Finally, while three hydrophilic residues, Ser102, Ser103, and Cys222, are identical in the GMPR family, the corresponding sites in IMPDHs contain hydrophobic residues.…”

“…12 While hGMPR1 and hGMPR2 do not have significant amino acid similarity to human type I IMPDH or type II IMPDH, 8 hGMPR2 does show similarity (31% identity in their deduced amino acid sequences) to the IMPDH of Borrelia burgdorferi. 13 The crystal structure of IMPDH has been solved for many species, [13][14][15][16][17][18] which share similarities in active sites and geometry structures. Recently, the crystal structure of hGMPR1 was released in the Protein Data Bank †.…”

The Functional Role of a Conserved Loop in EAL Domain-Based Cyclic di-GMP-Specific Phosphodiesterase

“…Despite the diverse reactions catalyzed by (␤/␣) 8 barrel fold enzymes, this extended loop, often referred to as loop 6, plays an important role as a functional lid for substrate sequestering, solvent exclusion, and product release (15). The loop was found to facilitate substrate binding and conformational transition in tryptophan synthase (3,4) and functions as a lid for substrate sequestering during catalysis in inosine 5Ј-monophosphate dehydrogenase (22). Notably, it was shown that the loop sways from the active site in the nonactive structure of ribulose-1,5-bisphosphate carboxylase but folds over to shield the active site from the solvent in the activated structure (21).…”