The enzyme inosine monophosphate dehydrogenase (IMPDH) catalyzes the unique initial step in guanine nucleotide synthesis. The active site of IMPDH has two binding pockets, one for the cofactor, nicotinamide adenine dinucleotide (NAD) and one for the substrate, inosine monophosphate (IMP). IMPDH is dependent on the NAD cofactor and binds IMP to produce xanthine monophosphate (XMP). Next in the pathway, XMP is aminated to guanosine monophosphate (GMP) which subsequently gives rise to one of the building blocks of DNA (dGTP) and RNA synthesis (GTP). A summary of the reaction is pictured below in Figure 1

Enzymes involved in nucleotide biosynthesis are essential for supporting cell proliferation. IMPDH is unregulated in rapidly dividing tumor cells and has therefore been identified as an excellent target for pharmacological intervention by several studies. In this study, we applied sequence-based bioinformatics to elucidate both the structural and functional characteristics of IMPDH. Specifically, a multiple sequence alignment of 78 IMPDH sequences was constructed and the program MEME was used to identify the most highly conserved motifs within the enzyme family. A phylogenetic tree analysis was performed which facilitated the separation of sequence into groups and the identification of distinguishing characteristics. Visualization of the conserved residues and motifs within a 3-dimensional atomic model of human IMPDH revealed the crucial interactions for binding both the cofactor and substrate, maintaining the structure of the catalytic core and for catalyzing the production of xanthine monophosphate from inosine monophosphate.

Structural and Functional Analysis of Inosine Monophosphate Dehydrogenase using Sequence-Based Bioinformatics

Barry Sexton and Troy Wymore Pittsburgh Supercomputing Center, Biomedical Initiative Group, Pittsburgh, PA 15213

Biomedical Initiative Group http://www.psc.edu/biomed

67940681|r

34897074|r21618070|g

4468193|em

28413147|g

66508366|r58377269|r

54643860|g

24641073|r

61823779|r

62859741|r

28422466|g

1708472|sp

34328209|r106722|pir25014074|s

28422324|g

60098399|e

38014713|g31981382|r

307066|gb|

13543973|g

28422611|g51703854|g

4104430|gb

56526128|e

23478193|g56498720|e

2239243|em6467900|gb

57222905|g

7920698|gb

58119409|g

6322012|re

577140|emb

66847125|g32420219|r

38105711|g

27360004|g

56708374|r

33595927|r

17428444|e

67549470|r

67666170|r 59718108|g7226438|gb

53802426|r

28199329|r

15839021|r21231623|r

58581817|r21108532|g

67675369|r

67156483|r

24982460|g

66044509|r 28868656|r

66857260|r

57242158|r

57167939|r

57237943|r6968494|em

15607007|r

62738151|p15644099|r

51894052|r19714854|g

29377734|r

52145201|r11251771|p

47525264|r

49476689|r

56418544|r

52078500|r

39959|emb|

16077077|r28204441|g

15025734|g

18145941|d

Figure 1. Inosine Monophosphate Dehydrogenase with highlighted motifs identified by MEME. The above figure shows just one monomer of IMPDH while the bottom figure shows the enzyme as a tetramer, the naturally occurring state.

Abstract/Methods

Figure 2. Global view of multiple sequence alignment in GeneDoc with highlighted motifs

Conclusions

Acknowledgments

Figure 3. Phylogenetic tree representation of 78 IMPDH sequences analyzed.

Mammal/Amphibian Mammal/Amphibian

InProtists

Fungi

Pla

Prokaryote I

Prokaryote II

Prokaryote III

Figure 4. Simplified representation of the phylogenetic tree revealing the major groups of IMPDH and their evolutionary relationships.

Figure 5. Interaction between residue cysteine 331 (yellow) and the inosine monophosphate substrate (green).

Figure 6. Interactions between the NAD cofactor (tan) and key residues asparagine 274, serine 275, serine 276, and phenylalanine 282 (red).

Figure 7. Residues (blue) which appear to interact with both the substrate (green) and the NAD cofactor (tan). Pictured residues are asparagine 303, arginine 322, and isoleucine 332.

NAD cofactor binding

Gly451 Leu460 Gly463 Hsd466

Yellow

Motif 9 - Residues 436 to 466 KIKVAQGVSGAVQDK GSIHKFVPYLIAGIQH

NAD cofactor binding

Tyr411 Met414 Arg429

Purple

Motif 8 - Residues 401 to 429 FFSDGIRLKKYRGMG SLDAMDKHLSSQNR

Inosine substrate binding and chemistry

Asp364 Gly365 Gly366 Ile367

Ser388 Leu389

Blue

Motif 7 - Residues 359 to 399 VPVIADGGIQNVGHIAKALALG

ASTVMMGSLLAATTEAPGE

NAD cofactor and inosine substrate

chemistry

Asn303 Arg322 Ile330 Cys331 Ile332 Glu335

Red

Motif 6 - Residues 294 to 343 YPNLQVIGGNVVTAAQAKNLIDAGVDAL

RVGMGSGSICITQEVLACGRPQ

NAD cofactor binding and chemistry

Asp274 Ser275 Ser276 Ser280

Phe282

Ice Blue

Motif 5 - Residues 263 to 291 LAQAGVDVVVLDSS QGNSIFQINMIKYIK

Structural

Leu218 Asp226 Lys228 Pro234

Cyan

Motif 4 - Residues 194 to 234 LKEANEILQRSKKGKLPIVNE DDELVAIIARTDLKKNRDYP

Structural

Asp117 Pro118 Pro123 Gly148

Pink

Motif 3 - Residues 114 to 163 FITDPVVLSPKDRVRDVFEAKARHG

FCGIPITDTGRMGSRLVGIISSRDI

NAD cofactor binding

Met70 Asp71 Thr72 Val73

His93

Orange

Motif 2 - Residues 64 to 113 PLVSSPMDTVTEAGMAIAMALTGGIGFI

HHNCTPEFQANEVRKVKKYEQG

NAD cofactor binding

Thr45 Ala46

Green

Motif 1 - Residues 29 to 57 GLTYNDFLILPGYIDFTADQ

VDLTSALTKKITLKTPLY

Possible Function

Crucial Residues

Color (Figure 1)

Motif & Consensus Sequence

● Bioengineering and Bioinformatics Summer Institute, Department of Computational Biology, University of Pittsburgh ● National Institute of Health (NIH) and National Science Foundation (NSF) ● Bucknell University, Department of Biology ● Troy Wymore, Pittsburgh Supercomputing Center, Biomedical Initiative Group ● Adam Marko, University of Pittsburgh ● Duquesne University

The constructed phylogenetic tree reveals the relative similarity among all 78 IMPDH sequences analyzed. In the simplified tree, a general development from prokaryotes to more advanced organisms such as protists and plants to the most developed organisms such as humans and other mammals conveys a probable evolutionary progression. IMPDH from prokaryotes as opposed to IMPDH from higher organisms contains an additional tenth conserved motif near the end of the chain, suggesting an inversion or substitution somewhere during the transition from bacteria to higher organisms in evolutionary time. Detailed information describing each identified motif, it’s most crucial residues, and it’s probable role can be found in the displayed table. Visualization and analysis of the conserved motifs and their key residues which interact with both the IMP substrate and NAD cofactor provide invaluable insight into the catalytic activity of IMPDH. Additionally, Information about key interactions between certain residues and the substrate and cofactor can enable biochemists to design a drug which would effectively bind and inhibit the enzyme. An efficient inhibitor of IMPDH would not only have applications to cancer therapy, but also to antiparasitic and antiviral agents.

(gram positive bacteria, pathogenic,

obligate aerobes)

(proteobacteria, pathogenic, gram

negative)

(rod shaped bacteria, gram positive, thermophylic

(budding yeasts, fission yeasts,

bread molds, etc.)

(pathogenic protozoa)

(flowering plants, rice,

tobacco, etc.)

(honey bee, mosquito, fruit

fly, etc.)

(human, mouse, chicken, frog, etc.) (human, mouse,

chicken, frog, etc.)