Structure and Activity Analyses of Escherichia coli K-12 NagD Provide Insight into the Evolution of Biochemical Function in the Haloalkanoic Acid Dehalogenase Superfamily^,

Abstract

The HAD superfamily is a large superfamily of proteins which share a conserved core domain that provides those active site residues responsible for the chemistry common to all family members. The superfamily is further divided into the four subfamilies I, IIA, IIB, and III, based on the topology and insertion site of a cap domain that provides substrate specificity. This structural and functional division implies that members of a given HAD structural subclass may target substrates that have similar structural characteristics. To understand the structure/function relationships in all of the subfamilies, a type IIA subfamily member, NagD from Escherichia coli K-12, was selected (type I, IIB, and III members have been more extensively studied). The structure of the NagD protein was solved to 1.80 A with R(work) = 19.8% and R(free) = 21.8%. Substrate screening and kinetic analysis showed NagD to have high specificity for nucleotide monophosphates with k(cat)/K(m) = 3.12 x 10(4) and 1.28 x 10(4) microM(-)(1) s(-)(1) for UMP and GMP, respectively. This specificity is consistent with the presence of analogues of NagD that exist as fusion proteins with a nucleotide pyrophosphatase from the Nudix family. Docking of the nucleoside substrate in the active site brings it in contact with conserved residues from the cap domain that can act as a substrate specificity loop (NagD residues 144-149) in the type IIA subfamily. NagD and other subfamily IIA and IIB members show the common trait that substrate specificity and catalytic efficiencies (k(cat)/K(m)) are low (1 x 10(4) M(-)(1) s(-)(1)) and the boundaries defining physiological substrates are somewhat overlapping. The ability to catabolize other related secondary metabolites indicates that there is regulation at the genetic level.

Discovery of a glycerol 3-phosphate phosphatase reveals glycerophospholipid polar head recycling in Mycobacterium tuberculosis

“…HADSF phosphatases act on chemically diverse substrates. Substrate specificity is determined by a structural insertion in the core, called a cap domain (10)(11)(12)(13)(14), which forms a lid over the active site cavity during catalysis. Rv1692 is a potential HADSF member conserved in mycobacteria.…”

“…The subfamily II cap is located between motifs II and III of the core domain and consists of two different α/β folds, designated types IIA and IIB. Contrary to the other two subfamilies, subfamily III does not possess a cap domain and has only a core domain with a connecting loop serving in place of the cap domain (13).…”

The catalytic scaffold of the haloalkanoic acid dehalogenase enzyme superfamily acts as a mold for the trigonal bipyramidal transition state

“…The essential nature of the role of Asp-10 in transition-state stabilization is supported by the finding that in all HADSF subfamilies, the equivalent residue (the second D of the DXD motif conserved among HAD phosphatases and phosphomutases) is itself positioned by a salt-bridge or hydrogen bond formed with a residue that is conserved in role, but not in identity (4,34). In HPP this residue, Arg-45 forms a salt bridge with Asp-10.…”

Functional Diversity of Haloacid Dehalogenase Superfamily Phosphatases from Saccharomyces cerevisiae

“…2). Previously, pronounced substrate promiscuity has been demonstrated for HAD phosphatases from E. coli and other organisms (28,32,40). This broad substrate specificity apparently contributes to the key role of these enzymes in the hydrolysis of a broad range of phosphomonoesters as well as the "house-cleaning" functions.…”

“…To date, many HAD-like phosphatases from different organisms have been characterized both biochemically and structurally, including the phosphoserine phosphatase SerB from Methanococcus jannaschii (30), phosphoglycolate phosphatase TA0175 from Thermoplasma acidophilum (31), UMP nucleotidase NagD from E. coli (32), and inorganic pyrophosphatase BT2127 from Bacteroides thetaiotaomicron (33). In particular, phosphoprotein phosphatase activity has been demonstrated for several eukaryotic HAD-like hydrolases: human CTDP1 (or FCP1; Ser(P) phosphatase) (34), Drosophila and mammalian Eyes absent (Tyr(P) phosphatase) (35)(36)(37), and mammalian chronophin (Ser(P) phosphatase) (38,39).…”