Anthony Addlagatta

Structure of aminopeptidase N from Escherichia coli suggests a compartmentalized, gated active site

Aminopeptidase N from Escherichia coli is a major metalloprotease that participates in the controlled hydrolysis of peptides in the proteolytic pathway. Determination of the 870-aa structure reveals that it has four domains similar to the tricorn-interacting factor F3. The thermolysin-like active site is enclosed within a large cavity with a volume of 2,200 Å3, which is inaccessible to substrates except for a small opening of approximately 8–10 Å. The substrate-based inhibitor bestatin binds to the protein with minimal changes, suggesting that this is the active form of the enzyme. The previously described structure of F3 had three distinct conformations that were described as “closed,” “intermediate,” and “open.” The structure of aminopeptidase N from E. coli, however, is substantially more closed than any of these. Taken together, the results suggest that these proteases, which are involved in intracellular peptide degradation, prevent inadvertent hydrolysis of inappropriate substrates by enclosing the active site within a large cavity. There is also some evidence that the open form of the enzyme, which admits substrates, remains inactive until it adopts the closed form.

Structural basis for the unusual specificity of Escherichia coli aminopeptidase N.

Aminopeptidase N from Escherichia coli is a M1 class aminopeptidase with the active-site region related to that of thermolysin. The enzyme has unusual specificity, cleaving adjacent to the large, nonpolar amino acids Phe and Tyr but also cleaving next to the polar residues Lys and Arg. To try to understand the structural basis for this pattern of hydrolysis, the structure of the enzyme was determined in complex with the amino acids L-arginine, L-lysine, L-phenylalanine, L-tryptophan, and L-tyrosine. These amino acids all bind with their backbone atoms close to the active-site zinc ion and their side chain occupying the S1 subsite. This subsite is in the form of a cylinder, about 10 A in cross-section and 12 A in length. The bottom of the cylinder includes the zinc ion and a number of polar side chains that make multiple hydrogen-bonding and other interactions with the alpha-amino group and the alpha-carboxylate of the bound amino acid. The walls of the S1 cylinder are hydrophobic and accommodate the nonpolar or largely nonpolar side chains of Phe and Tyr. The top of the cylinder is polar in character and includes bound water molecules. The epsilon-amino group of the bound lysine side chain and the guanidinium group of arginine both make multiple hydrogen bonds to this part of the S1 site. At the same time, the hydrocarbon part of the lysine and arginine side chains is accommodated within the nonpolar walls of the S1 cylinder. This combination of hydrophobic and hydrophilic binding surfaces explains the ability of ePepN to cleave Lys, Arg, Phe, and Tyr. Another favored substrate has Ala at the P1 position. The short, nonpolar side chain of this residue can clearly be bound within the hydrophobic part of the S1 cylinder, but the reason for its facile hydrolysis remains uncertain.

Elucidation of the function of type 1 human methionine aminopeptidase during cell cycle progression

Processing of the N-terminal initiator methionine is an essential cellular process conserved from prokaryotes to eukaryotes. The enzymes that remove N-terminal methionine are known as methionine aminopeptidases (MetAPs). Human MetAP2 has been shown to be required for the proliferation of endothelial cells and angiogenesis. The physiological function of MetAP1, however, has remained elusive. In this report we demonstrate that a family of inhibitors with a core structure of pyridine-2-carboxylic acid previously developed for the bacterial and yeast MetAP1 is also specific for human MetAP1 (HsMetAP1), as confirmed by both enzymatic assay and high-resolution x-ray crystallography. Treatment of tumor cell lines with the MetAP1-specific inhibitors led to an accumulation of cells in the G2/M phase, suggesting that HsMetAP1 may play an important role in G2/M phase transition. Overexpression of HsMetAP1, but not HsMetAP2, conferred resistance of cells to the inhibitors, and the inhibitors caused retention of N-terminal methionine of a known MetAP substrate, suggesting that HsMetAP1 is the cellular target for the inhibitors. In addition, when HsMetAP1 was knocked down by gene-specific siRNA, cells exhibited slower progression during G2/M phase, a phenotype similar to cells treated with MetAP1 inhibitors. Importantly, MetAP1 inhibitors were able to induce apoptosis of leukemia cell lines, presumably as a consequence of their interference with the G2/M phase checkpoint. Together, these results suggest that MetAP1 plays an important role in G2/M phase of the cell cycle and that it may serve as a promising target for the discovery and development of new anticancer agents.

Structure/function aspects of human 3β-hydroxysteroid dehydrogenase

Separate genes encode the human type 1 (placenta, breast tumors, other peripheral tissues) and type 2 (gonad, adrenal) isoforms of 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta-HSD1, 3 beta-HSD2). Mutagenesis of 3 beta-HSD1 produced the Y154F, H156Y and K158Q mutant enzymes in the probable Y(154)-P-H(156)-S-K(158) catalytic motif. The H(156)Y mutant of the 3 beta-HSD1 created a chimera of the 3 beta-HSD2 motif (Y(154)-P-Y(156)-S-K(158)) in 3 beta-HSD1. The D241N, D257L, D258L and D265N mutants are in the potential isomerase site of the 3 beta-HSD1 enzyme. Homology modeling with UDP-galactose-4-epimerase predicted that Asp(36) in the Rossmann-fold domain is responsible for the NAD(H) specificity of human 3 beta-HSD1, and our D36A/K37R mutant tested that assignment. The H(156)Y mutant of the 3 beta-HSD1 enzyme shifted the substrate (DHEA) kinetics to the 14-fold higher K(m) value measured for the 3 beta-HSD2 activity. From Dixon analysis, epostane inhibited the 3 beta-HSD1 activity with 17-fold greater affinity compared to 3 beta-HSD2 and H(156)Y. The mutants of Tyr(154) and Lys(158) exhibited no dehydrogenase activity and appear to be catalytic 3 beta-HSD residues. The D257L and D258L mutations eliminated isomerase activity, suggesting that Asp(257) or Asp(258) may be catalytic residues for isomerase activity. The D36A/K37R mutant shifted the cofactor preference of both 3 beta-HSD and isomerase from NAD(H) to NADP(H). In addition to characterizing catalytic residues, these studies have identified the structural basis (His(156)) for an exploitable difference in the substrate and inhibition kinetics of 3 beta-HSD1 and 3 beta-HSD2. Hence, it may be possible to selectively inhibit human 3 beta-HSD1 to slow the growth of hormone-sensitive breast tumor cells and control placental steroidogenesis near term to prevent premature labor.

Determining Structure and Function of Steroid Dehydrogenase Enzymes by Sequence Analysis, Homology Modeling, and Rational Mutational Analysis

Abstract: The short‐chain oxidoreductase (SCOR) family of enzymes includes over 6,000 members identified in sequenced genomes. Of these enzymes, ∼300 have been characterized functionally, and the three‐dimensional crystal structures of ∼40 have been reported. Since some SCOR enzymes are steroid dehydrogenases involved in hypertension, diabetes, breast cancer, and polycystic kidney disease, it is important to characterize the other members of the family for which the biological functions are currently unknown and to determine their three‐dimensional structure and mechanism of action. Although the SCOR family appears to have only a single fully conserved residue, it was possible, using bioinformatics methods, to determine characteristic fingerprints composed of 30‐40 residues that are conserved at the 70% or greater level in SCOR subgroups. These fingerprints permit reliable prediction of several important structure‐function features including cofactor preference, catalytic residues, and substrate specificity. Human type 1 3β‐hydroxysteroid dehydrogenase isomerase (3β‐HSDI) has 30% sequence identity with a human UDP galactose 4‐epimerase (UDPGE), a SCOR family enzyme for which an X‐ray structure has been reported. Both UDPGE and 3‐HSDI appear to trace their origins back to bacterial 3α,20β‐HSD. Combining three‐dimensional structural information and sequence data on the 3α,20β‐HSD, UDPGE, and 3β‐HSDI subfamilies with mutational analysis, we were able to identify the residues critical to the dehydrogenase function of 3‐HSDI. We also identified the residues most probably responsible for the isomerase activity of 3β‐HSDI. We test our predictions by specific mutations based on sequence analysis and our structure‐based model.

Structure of the angiogenesis inhibitor ovalicin bound to its noncognate target, human Type 1 methionine aminopeptidase.

Methionine aminopeptidases (MetAPs) remove the initiator methionine during protein biosynthesis. They exist in two isoforms, MetAP1 and MetAP2. The anti‐angiogenic compound fumagillin binds tightly to the Type 2 MetAPs but only weakly to Type 1. High‐affinity complexes of fumagillin and its relative ovalicin with Type 2 human MetAP have been reported. Here we describe the crystallographic structure of the low‐affinity complex between ovalicin and Type 1 human MetAP at 1.1 Å resolution. This provides the first opportunity to compare the structures of ovalicin or fumagillin bound to a Type 1 and a Type 2 MetAP. For both Type 1 and Type 2 human MetAPs the inhibitor makes a covalent adduct with a corresponding histidine. At the same time there are significant differences in the alignment of the inhibitors within the respective active sites. It has been argued that the lower affinity of ovalicin and fumagillin for the Type 1 MetAPs is due to the smaller size of their active sites relative to the Type 2 enzymes. Comparison with the uncomplexed structure of human Type 1 MetAP indicates that there is some truth to this. Several active site residues have to move “outward” by 0.5 Å or so to accommodate the inhibitor. Other residues move “inward.” There are, however, other factors that come into play. In particular, the side chain of His310 rotates by 134° into a different position where (together with Glu128 and Tyr195) it coordinates a metal ion not seen at this site in the native enzyme.