Kinetic and structural analysis of human ALDH9A1

Abstract Aldehyde dehydrogenases (ALDHs) constitute a superfamily of NAD(P)+-dependent enzymes, which detoxify aldehydes produced in various metabolic pathways to the corresponding carboxylic acids. Among the 19 human ALDHs, the cytosolic ALDH9A1 has so far never been fully enzymatically characterized and its structure is still unknown. Here, we report complete molecular and kinetic properties of human ALDH9A1 as well as three crystal forms at 2.3, 2.9, and 2.5 Å resolution. We show that ALDH9A1 exhibits wide substrate specificity to aminoaldehydes, aliphatic and aromatic aldehydes with a clear preference for γ-trimethylaminobutyraldehyde (TMABAL). The structure of ALDH9A1 reveals that the enzyme assembles as a tetramer. Each ALDH monomer displays a typical ALDHs fold composed of an oligomerization domain, a coenzyme domain, a catalytic domain, and an inter-domain linker highly conserved in amino-acid sequence and folding. Nonetheless, structural comparison reveals a position and a fold of the inter-domain linker of ALDH9A1 never observed in any other ALDH so far. This unique difference is not compatible with the presence of a bound substrate and a large conformational rearrangement of the linker up to 30 Å has to occur to allow the access of the substrate channel. Moreover, the αβE region consisting of an α-helix and a β-strand of the coenzyme domain at the dimer interface are disordered, likely due to the loss of interactions with the inter-domain linker, which leads to incomplete β-nicotinamide adenine dinucleotide (NAD+) binding pocket.

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Alternative N-terminal regions of Drosophila myosin heavy chain II regulate communication of the purine binding loop with the essential light chain

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.014684 ◽

2020 ◽

Vol 295 (42) ◽

pp. 14522-14535

Author(s):

Marieke J. Bloemink ◽

Karen H. Hsu ◽

Michael A. Geeves ◽

Sanford I. Bernstein

Keyword(s):

Light Chain ◽

Catalytic Domain ◽

Binding Pocket ◽

Kinetic Properties ◽

Myosin Isoforms ◽

Wild Type ◽

Essential Light Chain ◽

Adp Release ◽

Communication Pathway ◽

Binding Loop

We investigated the biochemical and biophysical properties of one of the four alternative exon-encoded regions within the Drosophila myosin catalytic domain. This region is encoded by alternative exons 3a and 3b and includes part of the N-terminal β-barrel. Chimeric myosin constructs (IFI-3a and EMB-3b) were generated by exchanging the exon 3–encoded areas between native slow embryonic body wall (EMB) and fast indirect flight muscle myosin isoforms (IFI). We found that this exchange alters the kinetic properties of the myosin S1 head. The ADP release rate (k-D) in the absence of actin is completely reversed for each chimera compared with the native isoforms. Steady-state data also suggest a reciprocal shift, with basal and actin-activated ATPase activity of IFI-3a showing reduced values compared with wild-type (WT) IFI, whereas for EMB-3b these values are increased compared with wild-type (WT) EMB. In the presence of actin, ADP affinity (KAD) is unchanged for IFI-3a, compared with IFI, but ADP affinity for EMB-3b is increased, compared with EMB, and shifted toward IFI values. ATP-induced dissociation of acto-S1 (K1k+2) is reduced for both exon 3 chimeras. Homology modeling, combined with a recently reported crystal structure for Drosophila EMB, indicates that the exon 3–encoded region in the myosin head is part of the communication pathway between the nucleotide binding pocket (purine binding loop) and the essential light chain, emphasizing an important role for this variable N-terminal domain in regulating actomyosin crossbridge kinetics, in particular with respect to the force-sensing properties of myosin isoforms.

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Functional Diversification of Tripeptidyl Peptidase and Endopeptidase Sedolisins in Fungi

10.1101/167379 ◽

2017 ◽

Cited By ~ 1

Author(s):

Facundo Orts ◽

Arjen ten Have

Keyword(s):

Conformational Change ◽

Computational Analysis ◽

Catalytic Domain ◽

Binding Pocket ◽

List Type ◽

Functional Diversification ◽

Specificity Determining Positions ◽

Tripeptidyl Peptidase ◽

Endopeptidase Activity ◽

Α Helix

AbstractSedolisins are acid proteases that are related to the basic subtilisins. They have been identified in all three superkingdoms but are not ubiquitous, although fungi that secrete acids as part of their lifestyle can have up to six paralogs. Both tripeptidyl peptidase (TPP) and endopeptidase activity have been identified and it has been suggested that these correspond to separate subfamilies.We studied eukaryotic sedolisins by computational analysis. A maximum likelihood tree shows three major clades of which two contain only fungal sequences. One fungal clade contains all known TPPs whereas the other contains the endosedolisins. We identified four cluster specific inserts (CSIs) in endosedolisins, of which CSIs 1, 3 and 4 appear as solvent exposed according to structure modeling. Part of CSI2 is exposed but a short stretch forms a novel and partially buried α-helix that induces a conformational change near the binding pocket. We also identified a total of 12 specificity determining positions (SDPs) divided over three SDP sub-networks. The major SDP network contains eight directly connected SDPs and modeling of virtual mutants suggests a key role for the W307A or F307A substitution. This substitution is accompanied by a group of four SDPs that physically interact at the interface of the catalytic domain and the enzyme’s prosegment. Modeling of virtual mutants suggests these SDPs are indeed required to compensate the conformational change induced by CSI2 and the A307. The additional major network SDPs as well as the two small SDP networks appear to be linked to this major substitution, all together explaining the hypothesized functional diversification of fungal sedolisins.HighlightsThere are two sedolisin subfamilies in fungi: tripeptidyl peptidases and endopeptidasesFunctional Diversification of fungal sedolisins led to a conformational change in the pocketFunctional Diversification centers around buried SDP307SDP307 is aromatic in TPPs and Alanine in endosedolisinsAdditional SDPs affect the interaction between core and chaperone-like prosegment

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Characterization of Recombinant Human Coagulation Factor XFriuli

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650267 ◽

1996 ◽

Vol 75 (02) ◽

pp. 313-317 ◽

Cited By ~ 8

Author(s):

D J Kim ◽

A Girolami ◽

H L James

Keyword(s):

Catalytic Domain ◽

Coagulation Factor ◽

Molecular Size ◽

Binding Pocket ◽

Site Directed Mutagenesis ◽

Bleeding Tendency ◽

Factor X ◽

Amino Terminal ◽

Normal Plasma ◽

Plasma Factor

SummaryNaturally occurring plasma factor XFriuli (pFXFr) is marginally activated by both the extrinsic and intrinsic coagulation pathways and has impaired catalytic potential. These studies were initiated to obtain confirmation that this molecule is multi-functionally defective due to the substitution of Ser for Pro at position 343 in the catalytic domain. By the Nelson-Long site-directed mutagenesis procedure a construct of cDNA in pRc/CMV was derived for recombinant factor XFriuli (rFXFr) produced in human embryonic (293) kidney cells. The rFXFr was purified and shown to have a molecular size identical to that of normal plasma factor X (pFX) by gel electrophoretic, and amino-terminal sequencing revealed normal processing cleavages. Using recombinant normal plasma factor X (rFXN) as a reference, the post-translational y-carboxy-glutamic acid (Gla) and (β-hydroxy aspartic acid (β-OH-Asp) content of rFXFr was over 85% and close to 100%, respectively, of expected levels. The specific activities of rFXFr in activation and catalytic assays were the same as those of pFXFr. Molecular modeling suggested the involvement of a new H-bond between the side-chains of Ser-343 and Thr-318 as they occur in anti-parallel (3-pleated sheets near the substrate-binding pocket of pFXFr. These results support the conclusion that the observed mutation in pFXFr is responsible for its dysfunctional activation and catalytic potentials, and that it accounts for the moderate bleeding tendency in the homozygous individuals who possess this variant procoagulant.

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The Crystal Structure of Shikimate Dehydrogenase (AroE) Reveals a Unique NADPH Binding Mode

Journal of Bacteriology ◽

10.1128/jb.185.14.4144-4151.2003 ◽

2003 ◽

Vol 185 (14) ◽

pp. 4144-4151 ◽

Cited By ~ 32

Author(s):

Sheng Ye ◽

Frank von Delft ◽

Alexei Brooun ◽

Mark W. Knuth ◽

Ronald V. Swanson ◽

...

Keyword(s):

Catalytic Domain ◽

Binding Pocket ◽

Binding Mode ◽

Sequence Motif ◽

Binary Complex ◽

Shikimate Dehydrogenase ◽

Conserved Residues ◽

Conserved Sequence ◽

Nicotinamide Mononucleotide ◽

Reversible Reduction

ABSTRACT Shikimate dehydrogenase catalyzes the NADPH-dependent reversible reduction of 3-dehydroshikimate to shikimate. We report the first X-ray structure of shikimate dehydrogenase from Haemophilus influenzae to 2.4-Å resolution and its complex with NADPH to 1.95-Å resolution. The molecule contains two domains, a catalytic domain with a novel open twisted α/β motif and an NADPH binding domain with a typical Rossmann fold. The enzyme contains a unique glycine-rich P-loop with a conserved sequence motif, GAGGXX, that results in NADPH adopting a nonstandard binding mode with the nicotinamide and ribose moieties disordered in the binary complex. A deep pocket with a narrow entrance between the two domains, containing strictly conserved residues primarily contributed by the catalytic domain, is identified as a potential 3-dehydroshikimate binding pocket. The flexibility of the nicotinamide mononucleotide portion of NADPH may be necessary for the substrate 3-dehydroshikimate to enter the pocket and for the release of the product shikimate.

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DIRECTED EVOLUTION OF METABOLIC PATHWAYS IN MICROBIAL POPULATIONS. I. MODIFICATION OF THE ACID PHOSPHATASE pH OPTIMUM IN S. CEREVISIAE

Genetics ◽

10.1093/genetics/70.1.59 ◽

1972 ◽

Vol 70 (1) ◽

pp. 59-73 ◽

Cited By ~ 2

Author(s):

J C Francis ◽

P E Hansche

Keyword(s):

Acid Phosphatase ◽

Metabolic Pathways ◽

Experimental System ◽

Microbial Populations ◽

Kinetic Properties ◽

Mutational Event ◽

Ph Optimum ◽

Functional Changes ◽

Evolution Of Metabolic Pathways ◽

Wide Range

ABSTRACT An experimental system for directing the evolution of enzymes and metabolic pathways in microbial populations is proposed and an initial test of its power is provided.—The test involved an attempt to genetically enhance certain functional properties of the enzyme acid phosphatase in S. cerevisiae by constructing an environment in which the functional changes desired would be "adaptive". Naturally occurring mutations in a population of 109 cells were automatically and continuously screened, over 1,000 generations, for their effect on the efficiency (Km) and activity of acid phosphatase at pH 6, and for their effect on the efficiency of orthophosphate metabolism.—The first adaptation observed, M1, was due to a single mutational event that effected a 30% increase in the efficiency of orthophosphate metabolism. The second, M2, effected an adaptive shift in the pH optimum of acid phosphatase and an increase in its activity over a wide range of pH values (an increment of 60% at pH 6). M2 was shown to result from a single mutational event in the region of the acid phosphatase structural gene. The third, M3, effected cell clumping, an adaptation to the culture apparatus that had no effect on phosphate metabolism.—The power of this system for directing the evolution of enzymes and of metabolic pathways is discussed in terms of the kinetic properties of the experimental system and in terms of the results obtained.

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Structure–function analysis of water-soluble inhibitors of the catalytic domain of exotoxin A from Pseudomonas aeruginosa

Biochemical Journal ◽

10.1042/bj20041480 ◽

2005 ◽

Vol 385 (3) ◽

pp. 667-675 ◽

Cited By ~ 33

Author(s):

Susan P. YATES ◽

Patricia L. TAYLOR ◽

René JØRGENSEN ◽

Dana FERRARIS ◽

Jie ZHANG ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Diphtheria Toxin ◽

Catalytic Domain ◽

Competitive Inhibition ◽

Function Analysis ◽

Binding Pocket ◽

Ring Structure ◽

Water Soluble ◽

Exotoxin A ◽

Ic50 Values

The mono-ADPRT (mono-ADP-ribosyltransferase), Pseudomonas aeruginosa ETA (exotoxin A), catalyses the transfer of ADP-ribose from NAD+ to its protein substrate. A series of water-soluble compounds that structurally mimic the nicotinamide moiety of NAD+ was investigated for their inhibition of the catalytic domain of ETA. The importance of an amide locked into a hetero-ring structure and a core hetero-ring system that is planar was a trend evident by the IC50 values. Also, the weaker inhibitors have core ring structures that are less planar and thus more flexible. One of the most potent inhibitors, PJ34, was further characterized and shown to exhibit competitive inhibition with an inhibition constant Ki of 140 nM. We also report the crystal structure of the catalytic domain of ETA in complex with PJ34, the first example of a mono-ADPRT in complex with an inhibitor. The 2.1 Å (1 Å=0.1 nm) resolution structure revealed that PJ34 is bound within the nicotinamide-binding pocket and forms stabilizing hydrogen bonds with the main chain of Gly-441 and to the side-chain oxygen of Gln-485, a member of a proposed catalytic loop. Structural comparison of this inhibitor complex with diphtheria toxin (a mono-ADPRT) and with PARPs [poly(ADP-ribose) polymerases] shows similarity of the catalytic residues; however, a loop similar to that found in ETA is present in diphtheria toxin but not in PARP. The present study provides insight into the important features required for inhibitors that mimic NAD+ and their binding to the mono-ADPRT family of toxins.

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Crystal structure of microtubule affinity-regulating kinase 4 catalytic domain in complex with a pyrazolopyrimidine inhibitor

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x15024747 ◽

2016 ◽

Vol 72 (2) ◽

pp. 129-134 ◽

Cited By ~ 22

Author(s):

John S. Sack ◽

Mian Gao ◽

Susan E. Kiefer ◽

Joseph E. Myers ◽

John A. Newitt ◽

...

Keyword(s):

Crystal Structure ◽

Catalytic Domain ◽

Neurofibrillary Tangle ◽

Binding Pocket ◽

Atp Binding ◽

Activation Loop ◽

Serine Threonine Kinase ◽

Atp Binding Site ◽

Threonine Kinase ◽

Catalytic Loop

Microtubule-associated protein/microtubule affinity-regulating kinase 4 (MARK4) is a serine/threonine kinase involved in the phosphorylation of MAP proteins that regulate microtubule dynamics. Abnormal activity of MARK4 has been proposed to contribute to neurofibrillary tangle formation in Alzheimer's disease. The crystal structure of the catalytic and ubiquitin-associated domains of MARK4 with a potent pyrazolopyrimidine inhibitor has been determined to 2.8 Å resolution with anRworkof 22.8%. The overall structure of MARK4 is similar to those of the other known MARK isoforms. The inhibitor is located in the ATP-binding site, with the pyrazolopyrimidine group interacting with the inter-lobe hinge region while the aminocyclohexane moiety interacts with the catalytic loop and the DFG motif, forcing the activation loop out of the ATP-binding pocket.

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Crystal Structure of thePseudomonas aeruginosaBEL-1 Extended-Spectrum β-Lactamase and its Complex with Moxalactam and Imipenem

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00936-16 ◽

2016 ◽

pp. AAC.00936-16 ◽

Cited By ~ 1

Author(s):

Cecilia Pozzi ◽

Filomena De Luca ◽

Manuela Benvenuti ◽

Laurent Poirel ◽

Patrice Nordmann ◽

...

Keyword(s):

Carbonyl Oxygen ◽

Side Chain ◽

Kinetic Properties ◽

Monoclinic Crystal ◽

Crystal Forms ◽

Monoclinic Form ◽

Hydrophobic Cavity ◽

Class A ◽

Enzyme Complexes ◽

Small Hydrophobic

BEL-1 is an acquired class A ESBL found inP. aeruginosaclinical isolates from Belgium, which is divergent from other ESBLs (max. identity, 54% with GES-type enzymes). This enzyme is efficiently inhibited by clavulanate, imipenem and moxalactam. Crystals of BEL-1 were obtained at pH 5.6 and the structure of native BEL-1 was determined from orthorhombic and monoclinic crystal forms, at 1.60-Å and 1.48 Å resolution, respectively. By soaking native BEL-1 crystals, complexes with imipenem (monoclinic form, 1.79 Å res.) and moxalactam (orthorhombic form, 1.85 Å res.) were also obtained. In the acyl-enzyme complexes, imipenem and moxalactam differ by the position of the α-substituent and of the carbonyl oxygen (in or out the oxyanion hole). More surprisingly, the Ω-loop, which includes the catalytically-relevant residue Glu166, was found in different conformations in the various subunits, resulting in the Glu166 side chain to be rotated outwards the active site or even in displacements of its Cα atom up to approx. 10 Å. A BEL-1 variant showing the single Leu162Phe substitution (BEL-2) confers a higher level of resistance to CAZ, CTX and FEP and shows significantly lowerKMvalues as compared to those of BEL-1, especially with oxyiminocephalosporins. BEL-1 Leu162 is located at the beginning of the Ω-loop, and surrounded by Phe72, Leu139, Leu148 and Leu169 (contact distances, 3.8-4.0 Å). This small hydrophobic cavity could not reasonably accommodate the bulkier Phe162 found in BEL-2 without altering neighboring residues or the Ω-loop itself, thus likely causing an important alteration of the enzyme kinetic properties.

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The High Resolution Crystal Structure of the Human Tumor Suppressor Maspin Reveals a Novel Conformational Switch in the G-helix

Journal of Biological Chemistry ◽

10.1074/jbc.m412043200 ◽

2005 ◽

Vol 280 (23) ◽

pp. 22356-22364 ◽

Cited By ~ 50

Author(s):

Ruby H. P. Law ◽

James A. Irving ◽

Ashley M. Buckle ◽

Katya Ruzyla ◽

Marguerite Buzza ◽

...

Keyword(s):

Crystal Structure ◽

High Resolution ◽

Tumor Suppressor ◽

Physiological Role ◽

Human Tumor ◽

Salt Bridge ◽

Structural Data ◽

Crystal Forms ◽

Reactive Center ◽

Α Helix

Maspin is a serpin that acts as a tumor suppressor in a range of human cancers, including tumors of the breast and lung. Maspin is crucial for development, because homozygous loss of the gene is lethal; however, the precise physiological role of the molecule is unclear. To gain insight into the function of human maspin, we have determined its crystal structure in two similar, but non-isomorphous crystal forms, to 2.1- and 2.8-Å resolution, respectively. The structure reveals that maspin adopts the native serpin fold in which the reactive center loop is expelled fully from the A β-sheet, makes minimal contacts with the core of the molecule, and exhibits a high degree of flexibility. A buried salt bridge unique to maspin orthologues causes an unusual bulge in the region around the D and E α-helices, an area of the molecule demonstrated in other serpins to be important for cofactor recognition. Strikingly, the structural data reveal that maspin is able to undergo conformational change in and around the G α-helix, switching between an open and a closed form. This change dictates the electrostatic character of a putative cofactor binding surface and highlights this region as a likely determinant of maspin function. The high resolution crystal structure of maspin provides a detailed molecular framework to elucidate the mechanism of function of this important tumor suppressor.

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The structure of the colorectal cancer-associated enzyme GalNAc-T12 reveals how nonconserved residues dictate its function

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1902211116 ◽

2019 ◽

Vol 116 (41) ◽

pp. 20404-20410 ◽

Cited By ~ 7

Author(s):

Amy J. Fernandez ◽

Earnest James Paul Daniel ◽

Sai Pooja Mahajan ◽

Jeffrey J. Gray ◽

Thomas A. Gerken ◽

...

Keyword(s):

Colorectal Cancer ◽

Molecular Basis ◽

Catalytic Domain ◽

Binding Pocket ◽

Substrate Selectivity ◽

Peptide Substrate ◽

X Ray ◽

Biochemical Studies ◽

Cancer Mutations ◽

Insight Into

Polypeptide N-acetylgalactosaminyl transferases (GalNAc-Ts) initiate mucin type O-glycosylation by catalyzing the transfer of N-acetylgalactosamine (GalNAc) to Ser or Thr on a protein substrate. Inactive and partially active variants of the isoenzyme GalNAc-T12 are present in subsets of patients with colorectal cancer, and several of these variants alter nonconserved residues with unknown functions. While previous biochemical studies have demonstrated that GalNAc-T12 selects for peptide and glycopeptide substrates through unique interactions with its catalytic and lectin domains, the molecular basis for this distinct substrate selectivity remains elusive. Here we examine the molecular basis of the activity and substrate selectivity of GalNAc-T12. The X-ray crystal structure of GalNAc-T12 in complex with a di-glycosylated peptide substrate reveals how a nonconserved GalNAc binding pocket in the GalNAc-T12 catalytic domain dictates its unique substrate selectivity. In addition, the structure provides insight into how colorectal cancer mutations disrupt the activity of GalNAc-T12 and illustrates how the rules dictating GalNAc-T12 function are distinct from those for other GalNAc-Ts.

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