A path from primary protein sequence to ligand recognition

Abstract

A novel method to organize protein structural information based solely on sequence is presented. The method clusters proteins into families that correlate with the three-dimensional protein structure and the conformation of the bound ligands. This procedure was applied to nicotinamide adenine dinucleotide [NAD(P)]-utilizing enzymes to identify a total of 94 sequence families, 53 of which are structurally characterized. Each of the structurally characterized proteins within a sequence family correlates to a single protein fold and to a common bound conformation of NAD(P). A wide range of structural folds is identified that recognize NAD(P), including Rossmann folds and beta/alpha barrels. The defined sequence families can be used to identify the type and prevalence of NAD(P)-utilizing enzymes in the proteomes of sequenced organisms. The proteome of Mycobacterium tuberculosis was mined to generate a proteome-wide profile of NAD(P)-utilizing enzymes coded by this organism. This enzyme family comprises approximately 6% of the open reading frames, with the largest subgroup being the Rossmann fold, short-chain dehydrogenases. The preponderance of short-chain dehydrogenases correlates strongly with the phenotype of M. tuberculosis, which is characterized as having one of the most complex prokaryotic cell walls.

Conformational Diversity of Ligands Bound to Proteins

“…10 Comparing clusters of sequences of NAD(P)-utilising enzymes from the SWISS-PROT database 11 with the conformations of bound cofactors, the main result reported is that each sequence family binds NAD(P) molecules in conformations which cluster together. This is not surprising, since the identification of protein relationships from sequence alone implies that more than 30% of their residues are identical, which in turn implies that function is conserved, and hence the cofactor should be expected to bind in the same way.…”

Identifying determinants of NADPH specificity in Baeyer–Villiger monooxygenases

“…NAD(P)(H)‐dependent enzymes are ubiquitous, and, from a large number of X‐ray structures, more than 10 different NAD(P)(H) binding folds can be identified, of which the Rossmann‐fold is the most common [3]. A recent study of enzyme–NAD(P)(H) complexes showed that the binding fold also correlates with the conformation of the NAD(P)(H) ligand [4]. The affinity for NAD(P)(H) can range from very tight binding, as in nicotinoproteins, which use the nucleotides as a cofactor [5], to weak binding, as is the case for p ‐hydroxybenzoate hydroxylase, an enzyme that does not have a recognizable NAD(P)H‐binding domain [6].…”

Cofactor fingerprinting with STD NMR to characterize proteins of unknown function: identification of a rare cCMP cofactor preference

“…Application of cofactor fingerprinting with STD NMR to dehydrogenases We initially applied the cofactor fingerprinting with STD NMR approach to well-characterized proteins (dehydrogenases), then to a protein of unknown function, as part of a lar- ger functional proteomics project [12][13][14][15]. Dehydrogenases are an excellent gene family for applying functional proteomic methods, since they represent 3-6% of most proteomes, and are easily identified using bioinformatics tools [16,17]. But, it is often difficult to predict based on sequence whether there will be preference for NADH or for its 2 0 -phosphorylated form (NADPH).…”

“…Dehydrogenases are an excellent gene family for applying functional proteomic methods, since they represent 3-6% of most proteomes, and are easily identified using bioinformatics tools [16,17]. But, it is often difficult to predict based on sequence whether there will be preference for NADH or for its 2 0 -phosphorylated form (NADPH).…”