Structural studies of domain movement in active-site mutants of porphobilinogen deaminase from<i>Bacillus megaterium</i>

Guo, Jingxu; Erskine, P.T.; Coker, Alun R.; Wood, Steve; Cooper, J.B.

doi:10.1107/s2053230x17015436

Cited by 7 publications

(6 citation statements)

References 48 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The missing coordinates of the residues 60-78 (part of the active-site loop and adjoining region) were modeled based on the structure of AtHMBS [PDB ID code 4HTG (10)] using Modeler 9v8 (22). The missing residues at the N-terminal (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) and the C-terminal (residues 358-361) were also modeled.…”

Section: Methodsmentioning

confidence: 99%

“…Previous studies of the purified Escherichia coli and human enzymes demonstrated the critical importance of a covalently bound dipyrromethane (DPM) cofactor and the occurrence of stable enzyme substrate complexes in the formation of HMB (4,(6)(7)(8). To date, the X-ray crystal structures of E. coli (9), Arabidopsis thaliana (10), Bacillus megaterium (11,12), and human (13,14) HMBS (hHMBS) with their DPM cofactors have been reported. The crystal structures of the hHMBS housekeeping isoform are available in the Protein Data Bank (PDB) under ID codes 3EQ1 (13) and 3ECR (14).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Human hydroxymethylbilane synthase: Molecular dynamics of the pyrrole chain elongation identifies step-specific residues that cause AIP

Bung

Roy

Chen

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Hydroxymethylbilane synthase (HMBS), the third enzyme in the heme biosynthetic pathway, catalyzes the head-to-tail condensation of four molecules of porphobilinogen (PBG) to form the linear tetrapyrrole 1-hydroxymethylbilane (HMB). Mutations in human () cause acute intermittent porphyria (AIP), an autosomal-dominant disorder characterized by life-threatening neurovisceral attacks. Although the 3D structure of hHMBS has been reported, the mechanism of the stepwise polymerization of four PBG molecules to form HMB remains unknown. Moreover, the specific roles of each of the critical active-site residues in the stepwise enzymatic mechanism and the dynamic behavior of hHMBS during catalysis have not been investigated. Here, we report atomistic studies of HMB stepwise synthesis by using molecular dynamics (MD) simulations, mutagenesis, and in vitro expression analyses. These studies revealed that the hHMBS active-site loop movement and cofactor turn created space for the elongating pyrrole chain. Twenty-seven residues around the active site and water molecules interacted to stabilize the large, negatively charged, elongating polypyrrole. Mutagenesis of these active-site residues altered the binding site, hindered cofactor binding, decreased catalysis, impaired ligand exit, and/or destabilized the enzyme. Based on intermediate stages of chain elongation, R26 and R167 were the strongest candidates for proton transfer to deaminate the incoming PBG molecules. Unbiased random acceleration MD simulations identified R167 as a gatekeeper and facilitator of HMB egress through the space between the enzyme's domains and the active-site loop. These studies identified the specific active-site residues involved in each step of pyrrole elongation, thereby providing the molecular bases of the active-site mutations causing AIP.

show abstract

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Human hydroxymethylbilane synthase: Molecular dynamics of the pyrrole chain elongation identifies step-specific residues that cause AIP

Bung

Roy

Chen

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…PDB entries 5ov4, 5ov5 and 5ov6 are crystal structures that each involve a mutant of the catalytic aspartic acid (Asp82 in Bacillus megaterium numbering). Guo et al (2017) reported that 'the only mutant, D82E, which has the whole cofactor bound in a well ordered manner is catalytically active, while the other two (D82A and D82N) are not'.…”

Section: Commentary On the Role Of The Pdb Data Files In Structural And Functional Studies Of Hmbsmentioning

confidence: 99%

The crystal structures of the enzyme hydroxymethylbilane synthase, also known as porphobilinogen deaminase

Helliwell

2021

Acta Cryst Sect F

View full text Add to dashboard Cite

The enzyme hydroxymethylbilane synthase (HMBS; EC 4.3.1.8), also known as porphobilinogen deaminase, catalyses the stepwise addition of four molecules of porphobilinogen to form the linear tetrapyrrole 1-hydroxymethylbilane. Thirty years of crystal structures are surveyed in this topical review. These crystal structures aim at the elucidation of the structural basis of the complex reaction mechanism involving the formation of tetrapyrrole from individual porphobilinogen units. The consistency between the various structures is assessed. This includes an evaluation of the precision of each molecular model and what was not modelled. A survey is also made of the crystallization conditions used in the context of the operational pH of the enzyme. The combination of 3D structural techniques, seeking accuracy, has also been a feature of this research effort. Thus, SAXS, NMR and computational molecular dynamics have also been applied. The general framework is also a considerable chemistry research effort to understand the function of the enzyme and its medical pathologies in acute intermittent porphyria (AIP). Mutational studies and their impact on the catalytic reaction provide insight into the basis of AIP and are also invaluable for guiding the understanding of the crystal structure results. Future directions for research on HMBS are described, including the need to determine the protonation states of key amino-acid residues identified as being catalytically important. The question remains – what is the molecular engine for this complex reaction? Thermal fluctuations are the only suggestion thus far.

show abstract

“…Before the widespread prevalence of Bacillus subtilis , B. megaterium had been widely used in biochemical research due to its extensive metabolic capacity and physical properties conducive to biotechnology applications ( Hitchins et al, 1968 ; Elmerich and Aubert, 1971 ). The strain is a commercially available, nonpathogenic host, which has been used to produce various enzymes, such as penicillin amidase, amylase, amino acid dehydrogenase, and glucose dehydrogenase, as well as to produce recombinant proteins ( Lammers et al, 2004 ; Malten et al, 2005 ; Biedendieck et al, 2011 ; Grage et al, 2017 ; Guo et al, 2017 ). Moreover, B. megaterium can synthesize vitamin B 12 through an oxygen-independent adenosylcobalamin pathway ( Eppinger et al, 2011 ).…”

Section: Introductionmentioning

confidence: 99%

Isolation and Identification of Antibacterial Bioactive Compounds From Bacillus megaterium L2

Xie

Peng

et al. 2021

Front. Microbiol.

View full text Add to dashboard Cite

Bacterial metabolites exhibit a variety of biologically active compounds including antibacterial and antifungal activities. It is well known that Bacillus is considered to be a promising source of bioactive secondary metabolites. Most plant pathogens have an incredible ability to mutate and acquire resistance, causing major economic losses in the agricultural field. Therefore, it is necessary to use the natural antibacterial compounds in microbes to control plant pathogens. This study was conducted to investigate the bio-active compounds of Bacillus megaterium L2. According to the activity guidance of Agrobacterium tumefaciens T-37, Erwinia carotovora EC-1 and Ralstonia solanacearum RS-2, five monomeric compounds, including erucamide (1), behenic acid (2), palmitic acid (3), phenylacetic acid (4), and β-sitosterol (5), were fractionated and purified from the crude ethyl acetate extract of B. megaterium. To our knowledge, all compounds were isolated from the bacterium for the first time. To understand the antimicrobial activity of these compounds, and their minimum inhibitory concentrations (MICs) (range: 0.98∼500 μg/mL) were determined by the broth microdilution method. For the three tested pathogens, palmitic acid exhibited almost no antibacterial activity (>500 μg/mL), while erucamide had moderate antibacterial activity (MIC = 500 μg/mL). Behenic acid showed MICs of 250 μg/mL against T-37 and RS-2 strains with an antibacterial activity. β-sitosterol showed significant antimicrobial activity against RS-2. β-sitosterol showed remarkable antimicrobial activity against RS-2 with an MIC of 15.6 μg/mL. In addition, with the antimicrobial activity, against T-37 (62.5 μg/mL) and against EC-1 (125 μg/mL) and RS-2 (15.6 μg/mL) strains notably, phenylacetic acid may be interesting for the prevention and control of phytopathogenic bacteria. Our findings suggest that isolated compounds such as behenic acid, β-sitosterol, and phenylacetic acid may be promising candidates for natural antimicrobial agents.

show abstract

Structural studies of domain movement in active-site mutants of porphobilinogen deaminase fromBacillus megaterium

Cited by 7 publications

References 48 publications

Human hydroxymethylbilane synthase: Molecular dynamics of the pyrrole chain elongation identifies step-specific residues that cause AIP

Human hydroxymethylbilane synthase: Molecular dynamics of the pyrrole chain elongation identifies step-specific residues that cause AIP

The crystal structures of the enzyme hydroxymethylbilane synthase, also known as porphobilinogen deaminase

Isolation and Identification of Antibacterial Bioactive Compounds From Bacillus megaterium L2

Contact Info

Product

Resources

About