Azzmer Azzar Abdul Hamid

Molecular Modelling and Functional Studies of the Non-Stereospecific α-Haloalkanoic Acid Dehalogenase (DehE) from Rhizobium SP. RC1 and its Association with 3-Chloropropionic Acid (β-Chlorinated Aliphatic Acid)

ABSTRACT Many environmental pollutions are caused by the abundance of xenobiotic compounds in nature. For instance, halogenated compounds released from chemical industries were proven to be toxic and recalcitrant in the environment. However, haloalkanoic acid dehalogenases can catalyse the removal of halides from organic haloacids and thus have gained interest for bioremediation and synthesis of industrial chemicals. This study presents the first structural model and the key residues of the non-stereospecific haloalkanoic acid dehalogenase, DehE, from Rhizobium sp. RC1. The enzyme was built using a homology modelling technique; the structure of DehI from Pseudomonas putida PP3 was used as a template, because of its homology to DehE. The structure of DehE consists of only α-helices. Twelve conserved residues that line the active site were identified: Trp34, Ala36, Phe37, Asn114, Tyr117 Ala187, Ser188, Asp189, Tyr265, Phe268, Ile269, and Ile272. These residues are consistent with the residues found in the active site of DehI and D, L-DEX 113 from Pseudomonas sp. 113. Asp189 activates the water molecule as a nucleophile to attack the substrate chiral centre, which would result in an inversion of configuration of either D- or L-substrates. Both D- and L-substrates bind to and interact with the enzyme by hydrogen bonding with three residues, Trp34, Phe37, and Ser188. In addition, a putative tunnel was also identified that would provide a channel for the substrate to access the binding site. Based on computational analysis, DehE was proven to have the substrate affinity towards 3-chloropropionic acid (3CP)/β-chlorinated aliphatic acid, however, its dehalogenation process is far from clear. This DehE structural information will allow for rational design of non-stereospecific haloalkanoic acid dehalogenases in the future.

A review on non-stereospecific haloalkanoic acid dehalogenases

Haloalkanoic acid dehalogenases remove halides from organic haloacids and have potential as bioremediation agents. DehE from Rhizobium sp. RC1, DehI from Pseudomonas putida PP3 and D,LDEX 113 from Pseudomonas sp. 113 are non-stereospecific dehalogenases that invert the configurations of D- and L- carbons bound to a halogen. The kinetics of DehE has been partially characterized and brominated compounds have greater specificity constant values than do the corresponding chlorinated compounds. The sequence of DehE is similar to that of DehI; therefore, the two enzymes may have similar structures and functions. The three-dimensional structure of DehI is known and its reaction mechanism was inferred from its structure and a mutagenesis study of D,L-DEX 113. Aspartate residues at positions 189 and 194 in DehI and D,L-DEX 113 were predicted to be involved in catalysis. These residues activate a water molecule that directly attacks the chiral carbon. Because DehE and DehI are sequentially related, delineating the structure of DehE is important to ascertain if the catalytic residues and reaction mechanism are the same for both enzymes. A structural prediction, sequence-homology modeling and a site-directed mutagenesis study of DehE might help achieve this goal. Key words: Haloalkanoic acids, non-stereospecific dehalogenase, DehE, Rhizobium sp. RC1, enzyme kinetics, protein structure prediction, site-directed mutagenesis.

An S188V Mutation Alters Substrate Specificity of Non-Stereospecific α- Haloalkanoic Acid Dehalogenase E (DehE)

The non-stereospecific α-haloalkanoic acid dehalogenase E (DehE) degrades many halogenated compounds but is ineffective against β-halogenated compounds such as 3-chloropropionic acid (3CP). Using molecular dynamics (MD) simulations and site-directed mutagenesis we show here that introducing the mutation S188V into DehE improves substrate specificity towards 3CP. MD simulations showed that residues W34, F37, and S188 of DehE were crucial for substrate binding. DehE showed strong binding ability for D-2-chloropropionic acid (D-2CP) and L-2-chloropropionic acid (L-2CP) but less affinity for 3CP. This reduced affinity was attributed to weak hydrogen bonding between 3CP and residue S188, as the carboxylate of 3CP forms rapidly interconverting hydrogen bonds with the backbone amide and side chain hydroxyl group of S188. By replacing S188 with a valine residue, we reduced the inter-molecular distance and stabilised bonding of the carboxylate of 3CP to hydrogens of the substrate-binding residues. Therefore, the S188V can act on 3CP, although its affinity is less strong than for D-2CP and L-2CP as assessed by Km. This successful alteration of DehE substrate specificity may promote the application of protein engineering strategies to other dehalogenases, thereby generating valuable tools for future bioremediation technologies.

Identification of functional residues essential for dehalogenation by the non-stereospecific α-haloalkanoic acid dehalogenase from Rhizobium sp. RC1

The non‐stereospecific α‐haloalkanoic acid dehalogenase DehE from Rhizobium sp. RC1 catalyzes the removal of the halide from α‐haloalkanoic acid D,L‐stereoisomers and, by doing so, converts them into hydroxyalkanoic acid L,D‐stereoisomers, respectively. DehE has been extensively studied to determine its potential to act as a bioremediation agent, but its structure/function relationship has not been characterized. For this study, we explored the functional relevance of several putative active‐site amino acids by site‐specific mutagenesis. Ten active‐site residues were mutated individually, and the dehalogenase activity of each of the 10 resulting mutants in soluble cell lysates against D‐ and L‐2‐chloropropionic acid was assessed. Interestingly, the mutants W34 → A, F37 → A, and S188 → A had diminished activity, suggesting that these residues are functionally relevant. Notably, the D189 → N mutant had no activity, which strongly implies that it is a catalytically important residue. Given our data, we propose a dehalogenation mechanism for DehE, which is the same as that suggested for other non‐stereospecific α‐haloalkanoic acid dehalogenases. To the best of our knowledge, this is the first report detailing a functional aspect for DehE, and our results could help pave the way for the bioengineering of haloalkanoic acid dehalogenases with improved catalytic properties.

Insights into the stereospecificity of the d-specific dehalogenase from Rhizobium sp. RC1 toward D-and L-2-chloropropionate

Halogenated compounds are recalcitrant environmental pollutants prevalent in agricultural fields, waste waters and industrial by-products, but they can be degraded by dehalogenase-containing microbes. Notably, 2-haloalkanoic acid dehalogenases are employed to resolve optically active chloropropionates, as exemplified by the d-specific dehalogenase from Rhizobium sp. RCI (DehD), which acts on d-2-chloropropionate but not on its l-enantiomer. The catalytic residues of this dehalogenase responsible for its affinity toward d-2-chloropropionate have not been experimentally determined, although its three-dimensional crystal structure has been solved. For this study, we performed in silico docking and molecular dynamic simulations of complexes formed by this dehalogenase and d- or l-2-chloropropionate. Arg134 of the enzyme plays the key role in the stereospecific binding and Arg16 is in a position that would allow it to activate a water molecule for hydrolytic attack on the d-2-chloropropionate chiral carbon for release of the halide ion to yield l-2-hydroxypropionate. We propose that within the DehD active site, the NH group of Arg134 can form a hydrogen bond with the carboxylate of d-2-chloropropionate with a strength of ∼4 kcal/mol that may act as an acid–base catalyst, whereas, when l-2-chloropropionate is present, this bond cannot be formed. The significance of the present work is vital for rational design of this dehalogenase in order to confirm the involvement of Arg16 and Arg134 residues implicated in hydrolysis and binding of d-2-chloropropionate in the active site of d-specific dehalogenase from Rhizobium sp. RC1.

Comparative structural analysis of fruit and stem bromelain from Ananas comosus

Cysteine proteases in pineapple (Ananas comosus) plants are phytotherapeutical agents that demonstrate anti-edematous, anti-inflammatory, anti-thrombotic and fibrinolytic activities. Bromelain has been identified as an active component and as a major protease of A. comosus. Bromelain has gained wide acceptance and compliance as a phytotherapeutical drug. The proteolytic fraction of pineapple stem is termed stem bromelain, while the one presents in the fruit is known as fruit bromelain. The amino acid sequence and domain analysis of the fruit and stem bromelains demonstrated several differences and similarities of these cysteine protease family members. In addition, analysis of the modelled fruit (BAA21848) and stem (CAA08861) bromelains revealed the presence of unique properties of the predicted structures. Sequence analysis and structural prediction of stem and fruit bromelains of A. comosus along with the comparison of both structures provides a new insight on their distinct properties for industrial application.

Molecular characterization of monochloroacetate-degrading arthrobacter sp. Strain d2 isolated from universiti teknologi malaysia agricultural area

ABSTRACT Arthrobacter sp. strains D2 and D3 and Labrys sp. strain D1 capable of degrading 20 mM monochloroacetic acid (MCA) were isolated from soil contaminated with herbicides and pesticides. All three isolates were able to grow on MCA as the sole source of carbon and energy with concomitant chloride ion release in the growth medium (19 mM). Strains D2 and D3 (cells doubling time 7 ± 0.3 h) grew four times faster than D1 (26 ± 0.1 h). Strain D2 was then further investigated and could also grow in 10 mM of monobromoacetic acid (MBA), 2,2-dichloropropionic acid (2,2DCP), d,l-2-chloropropionic acid (D,L2CP), l-2-chloropropionic acid (L-2CP), d-2-chloropropionic acid (D-2CP), and glycolate as the sole sources of carbon and energy. Dehalogenase gene amplification using group I primers revealed a 410-bp polymerase chain reaction (PCR) product, but there was none using group II primers. The partial amino acid sequence analysis of group I DehD2 dehalogenase showed at least 32% identity to the corresponding regions of DehE, DhlIV, DehI, and D,L-DEX, with key amino acid residues Ser188, Ala187, and Asp189. These amino acid residues were involved in substrate binding and catalysis and were conserved in the partial amino acid sequence.

Effect of 2014 massive flood on well water qualities: A case study on Kelantan River basin, Malaysia

Abstract The effect of physical and biological qualities of wells after submergence was assessed following December 2014 flood in Kelantan. Studies were carried out on a total of 65 wells from 13 stations around Kelantan River basin in which the wells’ water were sampled for pH, total dissolved solid (TDS), turbidity and microbial contamination. About 95% of the well showed to be contaminated, 7 out of 65 samples (11.1%) showed TDS values >400 μS·cm−1; and 19 samples (29.2%) recorded turbidity beyond 7.0 NTU. Statistical non-parametric tests carried out on independent groups showed that the status of well contamination was neither determined by both degree of submergence nor by the geographical location. Also the physico-chemical parameters are independent of flood inundation. However, TDS and turbidity values changed based on geographical location, at p < 0.05. Well from estuary recorded higher TDS (241.2 μS·cm−1 ±159.5 SD) and turbidity (8.04 NTU ± 6.53 SD) compared to those from inner basin (TDS at 156.3 μS·cm−1± 88.9 SD; turbidity at 2.90 NTU ± 2.46 SD), respectively. The flood water had played significant role in the transmission of existing contaminant, and most of the wells were unsafe for drinking. We concluded that the degree of flood submergence does not necessarily determine the severity of the well contamination in Kelantan, but the existing contamination may exacerbate further the potential risk during post flood period.

Interaction of monomeric Ebola VP40 protein with a plasma membrane: A coarse-grained molecular dynamics (CGMD) simulation study

Ebola virus is a lipid-enveloped filamentous virus that affects human and non-human primates and consists of several types of protein: nucleoprotein, VP30, VP35, L protein, VP40, VP24, and transmembrane glycoprotein. Among the Ebola virus proteins, its matrix protein VP40 is abundantly expressed during infection and plays a number of critical roles in oligomerization, budding and egress from the host cell. VP40 exists predominantly as a monomer at the inner leaflet of the plasma membrane, and has been suggested to interact with negatively charged lipids such as phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylserine (PS) via its cationic patch. The hydrophobic loop at the C-terminal domain has also been shown to be important in the interaction between the VP40 and the membrane. However, details of the molecular mechanisms underpinning their interactions are not fully understood. This study aimed at investigating the effects of mutation in the cationic patch and hydrophobic loop on the interaction between the VP40 monomer and the plasma membrane using coarse-grained molecular dynamics simulation (CGMD). Our simulations revealed that the interaction between VP40 and the plasma membrane is mediated by the cationic patch residues. This led to the clustering of PIP2 around the protein in the inner leaflet as a result of interactions between some cationic residues including R52, K127, K221, K224, K225, K256, K270, K274, K275 and K279 and PIP2 lipids via electrostatic interactions. Mutation of the cationic patch or hydrophobic loop amino acids caused the protein to bind at the inner leaflet of the plasma membrane in a different orientation, where no significant clustering of PIP2 was observed around the mutated protein. This study provides basic understanding of the interaction of the VP40 monomer and its mutants with the plasma membrane.